Phototrophic Conditions Research Articles

Flux balance analysis reveals acetate metabolism modulates cyclic electron flow and alternative glycolytic pathways in Chlamydomonas reinhardtii.

Cells of the green alga Chlamydomonas reinhardtii cultured in the presence of acetate perform mixotrophic growth, involving both photosynthesis and organic carbon assimilation. Under such conditions, cells exhibit a reduced capacity for photosynthesis but a higher growth rate, compared to phototrophic cultures. Better understanding of the down regulation of photosynthesis would enable more efficient conversion of carbon into valuable products like biofuels. In this study, Flux Balance Analysis (FBA) and Flux Variability Analysis (FVA) have been used with a genome scale model of C. reinhardtii to examine changes in intracellular flux distribution in order to explain their changing physiology. Additionally, a reaction essentiality analysis was performed to identify which reaction subsets are essential for a given growth condition. Our results suggest that exogenous acetate feeds into a modified tricarboxylic acid (TCA) cycle, which bypasses the CO2 evolution steps, explaining increases in biomass, consistent with experimental data. In addition, reactions of the oxidative pentose phosphate and glycolysis pathways, inactive under phototrophic conditions, show substantial flux under mixotrophic conditions. Importantly, acetate addition leads to an increased flux through cyclic electron flow (CEF), but results in a repression of CO2 fixation via Rubisco, explaining the down regulation of photosynthesis. However, although CEF enhances growth on acetate, it is not essential—impairment of CEF results in alternative metabolic pathways being increased. We have demonstrated how the reactions of photosynthesis interconnect with carbon metabolism on a global scale, and how systems approaches play a viable tool in understanding complex relationships at the scale of the organism.

Comparison of pigment and proximate compositions of Tisochrysis lutea in phototrophic and mixotrophic cultures

Pigment contents and proximate compositions are important traits to evaluate the nutritional value of microalgae. The environmental condition and nutrient availability in the culture medium are hypothesised to regulate algal colour, nutritional composition and production. This study aimed to compare the pigment and proximate compositions in Tisochrysis lutea under phototrophic and mixotrophic conditions in an attempt to improve algal product quality. Algae were grown in the phototrophic and mixotrophic conditions at 50 μmol photons m−2 s−1 for 12 h daily, but 50 mM glycerol was added as a carbon source in the mixotrophic culture. The pigment contents in algae significantly increased in the mixotrophic condition, and the contents of chlorophylls a and c and carotenoids increased nearly 60 % compared with those in the phototrophic condition. The contents of saturated and polyunsaturated fatty acids in algae were significantly higher while the content of monounsaturated fatty acids was significantly lower in the mixotrophic condition than those in the phototrophic condition. Although the contents of protein, lipid and carbohydrate in T. lutea were not different, their overall production was significantly enhanced in the mixotrophic condition compared to the phototrophic condition. This study indicates that mixotrophic culture promotes pigment and proximate production and the change of fatty acid profile in algae depends on the addition of organic carbon to the culture medium.

A proposed aerobic granules size development scheme for aerobic granulation process

Aerobic granulation is increasingly used in wastewater treatment due to its unique physical properties and microbial functionalities. Granule size defines the physical properties of granules based on biomass accumulation. This study aims to determine the profile of size development under two physicochemical conditions. Two identical bioreactors namely Rnp and Rp were operated under non-phototrophic and phototrophic conditions, respectively. An illustrative scheme was developed to comprehend the mechanism of size development that delineates the granular size throughout the granulation. Observations on granules’ size variation have shown that activated sludge revolutionised into the form of aerobic granules through the increase of biomass concentration in bioreactors which also determined the changes of granule size. Both reactors demonstrated that size transformed in a similar trend when tested with and without illumination. Thus, different types of aerobic granules may increase in size in the same way as recommended in the aerobic granule size development scheme.

Comparison of N and P requirements of Isochrysis galbana under phototrophic and mixotrophic conditions

This study investigated the optimal N and P requirements for the growth of Isochrysis galbana under phototrophic and mixotrophic conditions. Algae were cultured in the f/2 basal medium modified with N and P concentrations. In the phototrophic condition, three N forms: sodium nitrate, ammonium sulphate and urea were tested at six N levels (0, 12.5, 25, 50, 100 and 200 mg N L−1) and P was tested at five levels (0, 1.3, 2.6, 5.2 and 10.4 mg P L−1). In the mixotrophic condition, the N or P modified f/2 basal medium was supplemented with 50 mM glycerol as the source of organic carbon. Growth was significantly influenced by the trophic conditions and N sources. The optimal N was 12.5–200 mg NO3-N L−1, 12.5–25 mg NH3-N L−1 or 12.5–50 mg urea-N L−1. The optimal P was 1.3–5.2 mg PO4-P L−1 for growth. In all treatments, the algal production in mixotrophy was over twofold higher than that in phototrophy. The maximal algal dry weight (235.7 mg L−1) and nutrient to algal biomass conversion efficiency (21.7 mg mg−1) were obtained in the cultures with urea as the N source under mixotrophy. The requirements of N and P were not different in phototrophy and mixotrophy, but dry weight production and nutrient conversion efficiency in mixotrophy were greater than in phototrophy, indicating that mixotrophic culture is more effective in the production of I. galbana.

Cyanobacterial biofuels: new insights and strain design strategies revealed by computational modeling.

BackgroundCyanobacteria are increasingly recognized as promising cell factories for the production of renewable biofuels and chemical feedstocks from sunlight, CO2, and water. However, most biotechnological applications of these organisms are still characterized by low yields. Increasing the production performance of cyanobacteria remains therefore a crucial step.ResultsIn this work we use a stoichiometric network model of Synechocystis sp. PCC 6803 in combination with CASOP and minimal cut set analysis to systematically identify and characterize suitable strain design strategies for biofuel synthesis, specifically for ethanol and isobutanol. As a key result, improving upon other works, we demonstrate that higher-order knockout strategies exist in the model that lead to coupling of growth with high-yield biofuel synthesis under phototrophic conditions. Enumerating all potential knockout strategies (cut sets) reveals a unifying principle behind the identified strain designs, namely to reduce the ratio of ATP to NADPH produced by the photosynthetic electron transport chain. Accordingly, suitable knockout strategies seek to block cyclic and other alternate electron flows, such that ATP and NADPH are exclusively synthesized via the linear electron flow whose ATP/NADPH ratio is below that required for biomass synthesis. The products of interest are then utilized by the cell as sinks for reduction equivalents in excess. Importantly, the calculated intervention strategies do not rely on the assumption of optimal growth and they ensure that maintenance metabolism in the absence of light remains feasible. Our analyses furthermore suggest that a moderately increased ATP turnover, realized, for example, by ATP futile cycles or other ATP wasting mechanisms, represents a promising target to achieve increased biofuel yields.ConclusionOur study reveals key principles of rational metabolic engineering strategies in cyanobacteria towards biofuel production. The results clearly show that achieving obligatory coupling of growth and product synthesis in photosynthetic bacteria requires fundamentally different intervention strategies compared to heterotrophic organisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0128-x) contains supplementary material, which is available to authorized users.

Nitric oxide-triggered remodeling of chloroplast bioenergetics and thylakoid proteins upon nitrogen starvation in Chlamydomonas reinhardtii.

Starving microalgae for nitrogen sources is commonly used as a biotechnological tool to boost storage of reduced carbon into starch granules or lipid droplets, but the accompanying changes in bioenergetics have been little studied so far. Here, we report that the selective depletion of Rubisco and cytochrome b6f complex that occurs when Chlamydomonas reinhardtii is starved for nitrogen in the presence of acetate and under normoxic conditions is accompanied by a marked increase in chlororespiratory enzymes, which converts the photosynthetic thylakoid membrane into an intracellular matrix for oxidative catabolism of reductants. Cytochrome b6f subunits and most proteins specifically involved in their biogenesis are selectively degraded, mainly by the FtsH and Clp chloroplast proteases. This regulated degradation pathway does not require light, active photosynthesis, or state transitions but is prevented when respiration is impaired or under phototrophic conditions. We provide genetic and pharmacological evidence that NO production from intracellular nitrite governs this degradation pathway: Addition of a NO scavenger and of two distinct NO producers decrease and increase, respectively, the rate of cytochrome b6f degradation; NO-sensitive fluorescence probes, visualized by confocal microscopy, demonstrate that nitrogen-starved cells produce NO only when the cytochrome b6f degradation pathway is activated.

Isolation, characterization, and validation of oleaginous, multi-trophic, and haloalkaline-tolerant microalgae for two-stage cultivation

Mass outdoor cultivation of microalgae for biofuels and co-products faces challenges of low lipid productivity, contamination, inefficient CO2 supply, and difficulties in harvesting. A two-stage cultivation process was developed to address these challenges. This involves culturing microalgae in a fermentor heterotrophically or photobioreactor mixotrophically as first-stage to rapidly obtain high cell densities for inoculating a phototrophic open-pond culture featuring high levels of NaHCO3, pH, and salinity as second-stage. A microalgae that is tolerant of these phototrophic conditions, can use organic carbon, and can prolifically produce oil is key to the success of such a two-stage process. Two oleaginous, haloalkaline-tolerant, and multi-trophic green microalgae from soda lakes were isolated, identified, and compared in this study using a multi-instrument approach as candidates for such process. A model triacylglyceride (TAG) was developed for rapid, non-destructive lipid quantitation using liquid-state 1H NMR. A two-stage cultivation system and a high pH-mediated auto-flocculation method were tested on the selected strain ALP2 with a 1L fermentor and 40L open-tank. In unoptimized conditions, the stain achieved a final biomass concentration of 0.978gDCW/L, lipid content of 39.78% DCW, and auto-flocculation harvesting efficiency of 64.1%.

Cell-specific gene expression in Anabaena variabilis grown phototrophically, mixotrophically, and heterotrophically.

BackgroundWhen the filamentous cyanobacterium Anabaena variabilis grows aerobically without combined nitrogen, some vegetative cells differentiate into N2-fixing heterocysts, while the other vegetative cells perform photosynthesis. Microarrays of sequences within protein-encoding genes were probed with RNA purified from extracts of vegetative cells, from isolated heterocysts, and from whole filaments to investigate transcript levels, and carbon and energy metabolism, in vegetative cells and heterocysts in phototrophic, mixotrophic, and heterotrophic cultures.ResultsHeterocysts represent only 5% to 10% of cells in the filaments. Accordingly, levels of specific transcripts in vegetative cells were with few exceptions very close to those in whole filaments and, also with few exceptions (e.g., nif1 transcripts), levels of specific transcripts in heterocysts had little effect on the overall level of those transcripts in filaments. In phototrophic, mixotrophic, and heterotrophic growth conditions, respectively, 845, 649, and 846 genes showed more than 2-fold difference (p < 0.01) in transcript levels between vegetative cells and heterocysts. Principal component analysis showed that the culture conditions tested affected transcript patterns strongly in vegetative cells but much less in heterocysts. Transcript levels of the genes involved in phycobilisome assembly, photosynthesis, and CO2 assimilation were high in vegetative cells in phototrophic conditions, and decreased when fructose was provided. Our results suggest that Gln, Glu, Ser, Gly, Cys, Thr, and Pro can be actively produced in heterocysts. Whether other protein amino acids are synthesized in heterocysts is unclear. Two possible components of a sucrose transporter were identified that were upregulated in heterocysts in two growth conditions. We consider it likely that genes with unknown function represent a larger fraction of total transcripts in heterocysts than in vegetative cells across growth conditions.ConclusionsThis study provides the first comparison of transcript levels in heterocysts and vegetative cells from heterocyst-bearing filaments of Anabaena. Although the data presented do not give a complete picture of metabolism in either type of cell, they provide a metabolic scaffold on which to build future analyses of cell-specific processes and of the interactions of the two types of cells.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-759) contains supplementary material, which is available to authorized users.

Effects of Cultivation Conditions and Media Compositions on Lipid Productivity of Native Microalga &lt;i&gt;Chlorella vulgaris&lt;/i&gt; U3-3

A native microalgae strain Chlorella vulgaris U3-3from Baimaxueshan Yunnan province has been isolated and identified as a potential feedstock for biodiesel production. The biomass production, lipid productivity, and fatty acid compositions of U3-3 are investigated and compared under phototrophic, heterotrophic and mixotrophic conditions with various media. U3-3 under phototrophic growth on BG11 medium shows higher cellular lipid content (44.6%) than those under heterotrophic and mixotrophic conditions. And U3-3 display its higher biomass production (5.55g/L) than those under phototrophic and heterotrophic conditions growing on optimized medium with supply 35g/L glucose under mixotrophic condition. However, Chlorella vulgaris U3-3 growing under heterotrophic condition with optimized medium of supply 35g/L glucose exhibited more than 7.26-fold and 1.05-fold increase in lipid productivity compared with that under phototrophic and mixotrophic conditions. Moreover, Chlorella vulgaris U3-3 has suitable fatty acid compositions with high saturated acid contents (28.76%) and favorable degree of unsaturation (92.86) growing under heterotrophic condition with optimized medium of supply 35g/L glucose, suggesting its potential as a biodiesel feedstock on this cultivation condition and medium composition.

Photoautotrophic production of D-lactic acid in an engineered cyanobacterium

BackgroundThe world faces the challenge to develop sustainable technologies to replace thousands of products that have been generated from fossil fuels. Microbial cell factories serve as promising alternatives for the production of diverse commodity chemicals and biofuels from renewable resources. For example, polylactic acid (PLA) with its biodegradable properties is a sustainable, environmentally friendly alternative to polyethylene. At present, PLA microbial production is mainly dependent on food crops such as corn and sugarcane. Moreover, optically pure isomers of lactic acid are required for the production of PLA, where D-lactic acid controls the thermochemical and physical properties of PLA. Henceforth, production of D-lactic acid through a more sustainable source (CO2) is desirable.ResultsWe have performed metabolic engineering on Synechocystis sp. PCC 6803 for the phototrophic synthesis of optically pure D-lactic acid from CO2. Synthesis of optically pure D-lactic acid was achieved by utilizing a recently discovered enzyme (i.e., a mutated glycerol dehydrogenase, GlyDH*). Significant improvements in D-lactic acid synthesis were achieved through codon optimization and by balancing the cofactor (NADH) availability through the heterologous expression of a soluble transhydrogenase. We have also discovered that addition of acetate to the cultures improved lactic acid production. More interestingly, 13C-pathway analysis revealed that acetate was not used for the synthesis of lactic acid, but was mainly used for synthesis of certain biomass building blocks (such as leucine and glutamate). Finally, the optimal strain was able to accumulate 1.14 g/L (photoautotrophic condition) and 2.17 g/L (phototrophic condition with acetate) of D-lactate in 24 days.ConclusionsWe have demonstrated the photoautotrophic production of D-lactic acid by engineering a cyanobacterium Synechocystis 6803. The engineered strain shows an excellent D-lactic acid productivity from CO2. In the late growth phase, the lactate production rate by the engineered strain reached a maximum of ~0.19 g D-lactate/L/day (in the presence of acetate). This study serves as a good complement to the recent metabolic engineering work done on Synechocystis 6803 for L-lactate production. Thereby, our study may facilitate future developments in the use of cyanobacterial cell factories for the commercial production of high quality PLA.

Cultivation ofIsochrysis galbanain Phototrophic, Heterotrophic, and Mixotrophic Conditions

This study compared the growth and biomass production of Isochrysis galbana under hetero-, mixo-, and phototrophic conditions using different organic carbon sources. The growth of I. galbana was inhibited in heterotrophy but was enhanced in mixotrophy compared to that in phototrophy. Subsequently, the influences of organic carbon and environmental factors (light and salinity) on the growth of I. galbana were further investigated. Algal dry weight increased as glycerol concentrations increased from 0 to 200 mmol and the highest algal production occurred at 50 mmol glycerol. At a range of light intensities of 25–200 μmol photons m−2 s−2, the highest algal growth rate occurred at 100 photons μmol m−2 s−2. The growth of I. galbana was significantly affected by photoperiod, and the maximal dry weight was obtained at 12 h light and 12 h dark. In the salinity test, I. galbana could grow in a wide range of salinities from 10 to 65‰, but the 35‰ salinity was optimal. This study suggests that the growth and production of I. galbana can be improved using mixotrophic culture at 50 mmol glycerol in 35‰ salinity.

Transcriptional regulation of the Rhodobacter capsulatus response regulator CtrA

The Rhodobacter capsulatus response regulator CtrA controls the expression of 227 genes, some of which are upregulated by both the phosphorylated and unphosphorylated forms of CtrA. Therefore, CtrA concentration alone, regardless of phosphorylation state, may determine expression of downstream genes, yet little is known about the regulation of ctrA in R. capsulatus. In this study we used a ctrA : : lacZ fusion plasmid to study the effects of medium composition, growth conditions and growth phase on R. capsulatus ctrA gene expression. These experiments indicate that ctrA expression is higher when cultures are grown in phototrophic (anaerobic) conditions compared with chemotrophic (aerobic) conditions, and is higher when grown in a minimal medium compared with a rich medium. We used several mutants to investigate possible regulatory pathways, and found that in R. capsulatus ctrA is not autoregulated but is regulated by a quorum-sensing system. The expression of ctrA increased as cell cultures moved through exponential phase and into stationary phase, with high levels of expression persisting long after culture turbidity plateaued. Although this growth phase-dependent pattern of expression was also observed in a quorum-sensing mutant, the magnitude of ctrA expression was about 50% of the wild-type strain at all phases. Furthermore, reduction of phosphate concentration in the growth medium decreased ctrA expression in a culture density-independent manner, whereas reduction of malic acid (carbon source) or ammonium (nitrogen source) concentration had no effect. The regulation of ctrA expression in R. capsulatus appears to require the coordination of multiple pathways involved in detecting a variety of environmental conditions.

Effect of the Light Regime and Phototrophic Conditions on Growth of the H2-producing Green Alga Chlamydomonas Reinhardtii

Development of the capacity to produce hydrogen economically from renewable energy resources is of critical importance to the future viability of that fuel. The inexpensive and widely available green alga Chlamydomonas reinhardtii has the ability to photosynthetically synthesise molecular hydrogen. Green algal hydrogen production does not generate any toxic or polluting bi-products and could potentially offer value-added products derived from algal biomass. The growth of dense and healthy algal biomass is a vital requirement for efficient hydrogen production. Algal cell density is principally limited by the illumination conditions of the algal culture and by the availability of key nutrients, including the sources of carbon, nitrogen, sulphur and phosphorus. In this study, the effect of different light regimes and carbon dioxide feeds on Chlamydomonas reinhardtii growth were investigated. The objective was to increasing the algal growth rate and the cell density, leading to enhanced biohydrogen production. State-of-the art photobioreactors were used to grow algal cultures, and to measure the pH and optical density of those cultures. Under mixotrophic growth conditions, using both acetate and carbon dioxide, increasing the carbon dioxide feed rate increased the optical density of the culture but reduced the growth rate. Under autotrophic growth conditions, with carbon dioxide as the only carbon source, a carbon dioxide feed with a partial pressure of circa 11% was determined to optimise both the algal growth rate and the optical density.

Plastid terminal oxidase 2 (PTOX2) is the major oxidase involved in chlororespiration in Chlamydomonas

By homology with the unique plastid terminal oxidase (PTOX) found in plants, two genes encoding oxidases have been found in the Chlamydomonas genome, PTOX1 and PTOX2. Here we report the identification of a knockout mutant of PTOX2. Its molecular and functional characterization demonstrates that it encodes the oxidase most predominantly involved in chlororespiration in this algal species. In this mutant, the plastoquinone pool is constitutively reduced under dark-aerobic conditions, resulting in the mobile light-harvesting complexes being mainly, but reversibly, associated with photosystem I. Accordingly, the ptox2 mutant shows lower fitness than wild type when grown under phototrophic conditions. Single and double mutants devoid of the cytochrome b(6)f complex and PTOX2 were used to measure the oxidation rates of plastoquinols via PTOX1 and PTOX2. Those lacking both the cytochrome b(6)f complex and PTOX2 were more sensitive to light than the single mutants lacking either the cytochrome b(6)f complex or PTOX2, which discloses the role of PTOX2 under extreme conditions where the plastoquinone pool is overreduced. A model for chlororespiration is proposed to relate the electron flow rate through these alternative pathways and the redox state of plastoquinones in the dark. This model suggests that, in green algae and plants, the redox poise results from the balanced accumulation of PTOX and NADPH dehydrogenase.

Major factors affecting isocitrate lyase activity in Rhodobacter capsulatus B10 under phototrophic conditions

External factors affecting the activity of isocitrate lyase (ICL) in Rhodobacter capsulatus B10 grown under controlled photoheterotrophic anaerobic conditions were investigated. The activity of this enzyme was found to depend on the history of the inoculum and on the growth phase on acetate medium. Intracellular degradation of ICL under unfavorable conditions was shown. However, transition of the growing culture from acetate to lactate did not result in active degradation of the enzyme. When transferred to acetate, Rba. capsulatus could grow without the lag phase and did not exhibit ICL activity, suggesting another anaplerotic pathway in Rba. capsulatus cells. Since emergence of the ICL activity in the cells grown on acetate results in an increase in its growth rate, the glyoxylate bypass plays an important role in acetate metabolism of Rba. Capsulatus.

IRsp1095: A genome-scale reconstruction of the Rhodobacter sphaeroides metabolic network

BackgroundRhodobacter sphaeroides is one of the best studied purple non-sulfur photosynthetic bacteria and serves as an excellent model for the study of photosynthesis and the metabolic capabilities of this and related facultative organisms. The ability of R. sphaeroides to produce hydrogen (H2), polyhydroxybutyrate (PHB) or other hydrocarbons, as well as its ability to utilize atmospheric carbon dioxide (CO2) as a carbon source under defined conditions, make it an excellent candidate for use in a wide variety of biotechnological applications. A genome-level understanding of its metabolic capabilities should help realize this biotechnological potential.ResultsHere we present a genome-scale metabolic network model for R. sphaeroides strain 2.4.1, designated iRsp1095, consisting of 1,095 genes, 796 metabolites and 1158 reactions, including R. sphaeroides-specific biomass reactions developed in this study. Constraint-based analysis showed that iRsp1095 agreed well with experimental observations when modeling growth under respiratory and phototrophic conditions. Genes essential for phototrophic growth were predicted by single gene deletion analysis. During pathway-level analyses of R. sphaeroides metabolism, an alternative route for CO2 assimilation was identified. Evaluation of photoheterotrophic H2 production using iRsp1095 indicated that maximal yield would be obtained from growing cells, with this predicted maximum ~50% higher than that observed experimentally from wild type cells. Competing pathways that might prevent the achievement of this theoretical maximum were identified to guide future genetic studies.ConclusionsiRsp1095 provides a robust framework for future metabolic engineering efforts to optimize the solar- and nutrient-powered production of biofuels and other valuable products by R. sphaeroides and closely related organisms.

Isolation and application of a wild strain photosynthetic bacterium to environmental waste management

A new photosynthetic bacterium isolate was morphologically identified as a non-motile rod-shape gram-negative bacterium. It produced a dark red culture under phototrophic condition, reproduced by budding and formed a lamellar intracytoplasmic membrane system parallel to cytoplasmic membrane, which contained bacteriochlorophyll a and caratenoids. It's physiological and nutrient requirement tests gave indication that the isolate thrived and multiplied in varied environmental conditions. It was consequently named Z08 and identified as Rhodobacter sphaeroides by 16SrDNA. Adaptation of Z08 to biodegradation of two environmentally concerned wastewaters, i.e. soybean and pharmaceutical wastewaters, attested its potential in wastewater bioremediation. Z08 adaptation in a suspended batch photobioreactor treating pharmaceutical wastewater at 3500lx radiation recorded best result after wastewater dilution of 1:4 with concomitant chemical oxygen demand reduction, biomass yield and specific growth of 50 %, 780 mg/L and 0.015/h, respectively at the lowest hydraulic retention time of three days. Furthermore, gas chromatography mass spectra analyses of treated and raw pharmaceutical wastewater indicated that high molecular weight recalcitrant compounds found in the pharmaceutical wastewater were transformed to less toxic and acceptable lower molecular weight substances through biodegradation. Whilst Z08 treatment of soybean wastewater under natural light intensity radiation recorded 80 % reduction, 1540 mg/L and 0.025/h for chemical oxygen demand, biomass and specific growth rate respectively regardless of the food to microorganism ratio. This preliminary investigation showed that isolate Z08 has some toxic tolerance level which could detoxify refractory substances with great potential for cell protein recovery in high organic strength wastewater. Therefore, strain Z08 could be employed in biodegradation of contaminated wastewater streams.

Coproporphyrin Excretion and Low Thiol Levels Caused by Point Mutation in the Rhodobacter sphaeroides S -Adenosylmethionine Synthetase Gene

A spontaneous mutant of Rhodobacter sphaeroides f. sp. denitrificans IL-106 was found to excrete a large amount of a red compound identified as coproporphyrin III, an intermediate in bacteriochlorophyll and heme synthesis. The mutant, named PORF, is able to grow under phototrophic conditions but has low levels of intracellular cysteine and glutathione and overexpresses the cysteine synthase CysK. The expression of molybdoenzymes such as dimethyl sulfoxide (DMSO) and nitrate reductases is also affected under certain growth conditions. Excretion of coproporphyrin and overexpression of CysK are not directly related but were both found to be consequences of a diminished synthesis of the key metabolite S-adenosylmethionine (SAM). The wild-type phenotype is restored when the gene metK encoding SAM synthetase is supplied in trans. The metK gene in the mutant strain has a mutation leading to a single amino acid change (H145Y) in the encoded protein. This point mutation is responsible for a 70% decrease in intracellular SAM content which probably affects the activities of numerous SAM-dependent enzymes such as coproporphyrinogen oxidase (HemN); uroporphyrinogen III methyltransferase (CobA), which is involved in siroheme synthesis; and molybdenum cofactor biosynthesis protein A (MoaA). We propose a model showing that the attenuation of the activities of SAM-dependent enzymes in the mutant could be responsible for the coproporphyrin excretion, the low cysteine and glutathione contents, and the decrease in DMSO and nitrate reductase activities.

Systems Analysis of Bioenergetics and Growth of the Extreme Halophile Halobacterium salinarum

Halobacterium salinarum is a bioenergetically flexible, halophilic microorganism that can generate energy by respiration, photosynthesis, and the fermentation of arginine. In a previous study, using a genome-scale metabolic model, we have shown that the archaeon unexpectedly degrades essential amino acids under aerobic conditions, a behavior that can lead to the termination of growth earlier than necessary. Here, we further integratively investigate energy generation, nutrient utilization, and biomass production using an extended methodology that accounts for dynamically changing transport patterns, including those that arise from interactions among the supplied metabolites. Moreover, we widen the scope of our analysis to include phototrophic conditions to explore the interplay between different bioenergetic modes. Surprisingly, we found that cells also degrade essential amino acids even during phototropy, when energy should already be abundant. We also found that under both conditions considerable amounts of nutrients that were taken up were neither incorporated into the biomass nor used as respiratory substrates, implying the considerable production and accumulation of several metabolites in the medium. Some of these are likely the products of forms of overflow metabolism. In addition, our results also show that arginine fermentation, contrary to what is typically assumed, occurs simultaneously with respiration and photosynthesis and can contribute energy in levels that are comparable to the primary bioenergetic modes, if not more. These findings portray a picture that the organism takes an approach toward growth that favors the here and now, even at the cost of longer-term concerns. We believe that the seemingly “greedy” behavior exhibited actually consists of adaptations by the organism to its natural environments, where nutrients are not only irregularly available but may altogether be absent for extended periods that may span several years. Such a setting probably predisposed the cells to grow as much as possible when the conditions become favorable.

Soluble proteome investigation of cobalt effect on the carotenoidless mutant ofRhodobacter sphaeroides

To investigate the surviving capability of Rhodobacter sphaeroides under phototrophic conditions in the presence of high cobalt concentration and its influence on the photosynthetic apparatus biosynthesis. Cells from R. sphaeroides strain R26.1 were grown anaerobically in a medium containing 5.0 mmol l(-1) cobalt ions and in a control medium. Metal toxicity was investigated comparing the soluble proteome of Co(2+)-exposed cells and cells grown in control medium by two-dimensional gel electrophoretic analysis. Significant changes in the expression level were detected for 43 proteins, the majority (35) being up-regulated. The enzyme porphobilinogen deaminase (PBGD) was found down-regulated and its activity was investigated. The up-regulated enzymes mainly belong to the general category of proteins and DNA degradation enzymes, suggesting that part of the catabolic reaction products can rescue bacterial growth in photosynthetically impaired cells. Furthermore, the down-regulation of PBGD strongly indicates that this key enzyme of the tetrapyrrole and bacteriochlorophyll synthesis is directly involved in the metabolic response. Data and experiments show that the cobalt detrimental effect on the photosynthetic growth of R. sphaeroides is associated with an impaired expression and functioning of PBGD.