Chlorophyll a Synthesis in Rhodobacter sphaeroides by Chlorophyll Synthase of Nicotiana tabacum.

The production of phytylated chlorophyll a (Chl aP) in Rhodobacter sphaeroides, which uses phytylated bacteriochlorophyll a (BChl aP), is the first step in expanding the light absorption spectra. Unlike the chlorophyll synthase (ChlG) of the Synechocystis sp. PCC6803, ChlGs of angiosperms, including Arabidopsis thaliana, Nicotiana tabacum, Avena sativa, and Oryza sativa, showed bacteriochlorophyll synthase activity and resistance to inhibition by bacteriochlorophyllide a (BChlide a), geranylgeranylated BChl a (BChl aGG), and BChl aP, collectively called bacteriochlorins. Among the angiosperm ChlGs, N. tabacum ChlG had the highest bacteriochlorophyll synthase activity and resistance to inhibition by bacteriochlorins. Expression of N. tabacum chlG in R. sphaeroides resulted in the formation of free Chl aP in the presence of BChl aP during photoheterotrophic growth, even though reactive oxygen species were generated.

The Photoactive Photosynthetic Reaction Center of a Rhodobacter sphaeroides Mutant Lacking 3-Vinyl (Bacterio)Chlorophyllide a Hydratase Contains 3-Vinyl Bacteriochlorophyll a.

Rhodobacter sphaeroides mutant BF-lacking 3-vinyl (bacterio)chlorophyllide a hydratase (BchF)-accumulates chlorophyllide a (Chlide a) and 3-vinyl bacteriochlorophyllide a (3V-Bchlide a). BF synthesizes 3-vinyl bacteriochlorophyll a (3V-Bchl a) through prenylation of 3V-Bchlide a and assembles a novel reaction center (V-RC) using 3V-Bchl a and Mg-free 3-vinyl bacteriopheophytin a (3V-Bpheo a) at a molar ratio of 2:1. We aimed to verify whether a bchF-deleted R. sphaeroides mutant produces a photochemically active RC that facilitates photoheterotrophic growth. The mutant grew photoheterotrophically-implying a functional V-RC-as confirmed by the emergence of growth-competent suppressors of bchC-deleted mutant (BC) under irradiation. Suppressor mutations in BC were localized to bchF, which diminished BchF activity and caused 3V-Bchlide a accumulation. bchF expression carrying the suppressor mutations in trans resulted in the coproduction of V-RC and wild-type RC (WT-RC) in BF. The V-RC had a time constant (τ) for electron transfer from the primary electron donor P (a dimer of 3V-Bchl a) to the A-side containing 3V-Bpheo a (HA) similar to that of the WT-RC and a 60% higher τ for electron transfer from HA to quinone A (QA). Thus, the electron transfer from HA to QA in the V-RC should be slower than that in the WT-RC. Furthermore, the midpoint redox potential of P/P+ of the V-RC was 33 mV more positive than that of the WT-RC. R. sphaeroides, thus, synthesizes the V-RC when 3V-Bchlide a accumulates. The V-RC can support photoheterotrophic growth; however, its photochemical activity is inferior to that of the WT-RC. IMPORTANCE 3V-Bchlide a is an intermediate in the bacteriochlorophyll a (Bchl a)-specific biosynthetic branch and prenylated by bacteriochlorophyll synthase. R. sphaeroides synthesizes V-RC that absorbs light at short wavelengths. The V-RC was not previously discovered because 3V-Bchlide a does not accumulate during the growth of WT cells synthesizing Bchl a. The levels of reactive oxygen species increased with the onset of photoheterotrophic growth in BF, resulting in a long lag period. Although the inhibitor of BchF is unknown, the V-RC may act as a substitute for the WT-RC when BchF is completely inhibited. Alternatively, it may act synergistically with WT-RC at low levels of BchF activity. The V-RC may broaden the absorption spectra of R. sphaeroides and supplement its photosynthetic ability at various wavelengths of visible light to a greater extent than that by the WT-RC alone.

Sustainability of in vitro light-dependent NADPH generation by the thylakoid membrane of Synechocystis sp. PCC6803.

BACKGROUND: NADPH is used as a reductant in various biosynthetic reactions. Cell-free bio-systems have gained considerable attention owing to their high energy utilization and time efficiency. Efforts have been made to continuously supply reducing power to the reaction mixture in a cyclical manner. The thylakoid membrane (TM) is a promising molecular energy generator, producing NADPH under light. Thus, TM sustainability is of major relevance for its in vitro utilization. RESULTS: Over 70% of TMs prepared from Synechocystis sp. PCC6803 existed in a sealed vesicular structure, with the F1 complex of ATP synthase facing outward (right-side-out), producing NADPH and ATP under light. The NADPH generation activity of TM increased approximately two-fold with the addition of carbonyl cyanide-p-(trifluoromethoxy) phenylhydrazone (FCCP) or removal of the F1 complex using EDTA. Thus, the uncoupling of proton translocation from the electron transport chain or proton leakage through the Fo complex resulted in greater NADPH generation. Biosilicified TM retained more than 80% of its NADPH generation activity after a week at 30°C in the dark. However, activity declined sharply to below 30% after two days in light. The introduction of engineered water-forming NADPH oxidase (Noxm) to keep the electron transport chain of TM working resulted in the improved sustainability of NADPH generation activity in a ratio (Noxm to TM)-dependent manner, which correlated with the decrease of singlet oxygen generation. Removal of reactive oxygen species (ROS) by catalase further highlighted the sustainable NADPH generation activity of up to 80% in two days under light. CONCLUSION: Reducing power generated by light energy has to be consumed for TM sustainability. Otherwise, TM can generate singlet oxygen, causing oxidative damage. Thus, TMs should be kept in the dark when not in use. Although NADPH generation activity by TM can be extended via silica encapsulation, further removal of hydrogen peroxide results in an improvement of TM sustainability. Therefore, as long as ROS formation by TM in light is properly handled, it can be used as a promising source of reducing power for in vitro biochemical reactions.

Photoautotrophic Growth Rate Enhancement of Synechocystis sp. PCC6803 by Heterologous Production of 2-Oxoglutarate:Ferredoxin Oxidoreductase from Chlorobaculum tepidum.

2-Oxoglutarate:ferredoxin oxidoreductase from Chlorobaculum tepidum (CtOGOR) is a carbon-fixing enzyme in the reductive TCA cycle that reversibly carboxylates succinyl-CoA to yield 2-oxoglutarate. CtOGOR is a heterotetramer of two large (α = 68 kDa) and two small (ß = 38 kDa) subunits. The αß protomer harbors one thiamine pyrophosphate and two 4Fe-4S clusters. Nonetheless, the enzyme has a considerable oxygen tolerance with a half-life of 143 min at 215 µM dissolved oxygen. Kinetic analyses of the purified recombinant CtOGOR revealed a lower Km for succinyl-CoA than for 2-oxoglutarate. Cellular levels of 2-oxoglutarate and glutamate­a product of glutamine oxoglutarate aminotransferase and glutamate dehydrogenase­increased more than twofold in the exponential phase compared with the control strain, leading to an approximately >30% increase in the photoautotrophic growth rate. Thus, CtOGOR was successfully produced in Synechocystis, thereby boosting carboxylation, resulting in enhanced photoautotrophic growth.

Production of long-chain free fatty acids from metabolically engineered Rhodobacter sphaeroides heterologously producing periplasmic phospholipase A2 in dodecane-overlaid two-phase culture.

BACKGROUND: Long-chain free fatty acids (FFAs) are a type of backbone molecule that can react with alcohol to produce biodiesels. Various microorganisms have become potent producers of FFAs. Efforts have focused on increasing metabolic flux to the synthesis of either neutral fat or fatty acyl intermediates attached to acyl carrier protein (ACP), which are the source of FFAs. Membrane lipids are also a source of FFAs. As an alternative way of producing FFAs, exogenous phospholipase may be used after heterologous production and localization in the periplasmic space. In this work, we examined whether Rhodobacter sphaeroides, which forms an intracytoplasmic membrane, can be used for long-chain FFA production using phospholipase. RESULTS: The recombinant R. sphaeroides strain Rs-A2, which heterologously produces Arabidopsis thaliana phospholipase A2 (PLA2) in the periplasm, excretes FFAs during growth. FFA productivity under photoheterotrophic conditions is higher than that observed under aerobic or semiaerobic conditions. When the biosynthetic enzymes for FA (ß-ketoacyl-ACP synthase, FabH) and phosphatidate (1-acyl-sn-glycerol-3-phosphate acyltransferase, PlsC) were overproduced in Rs-A2, the FFA productivity of the resulting strain Rs-HCA2 was elevated, and the FFAs produced mainly consisted of long-chain FAs of cis-vaccenate, stearate, and palmitate in an approximately equimolar ratio. The high-cell-density culture of Rs-HCA2 with DMSO in two-phase culture with dodecane resulted in an increase of overall carbon substrate consumption, which subsequently leads to a large increase in FFA productivity of up to 2.0 g L-1 day-1. Overexpression of the genes encoding phosphate acyltransferase (PlsX) and glycerol-3-phosphate acyltransferase (PlsY), which catalyze the biosynthetic steps immediately upstream from PlsC, in Rs-HCA2 generated Rs-HXYCA2, which grew faster than Rs-HCA2 and showed an FFA productivity of 2.8 g L-1 day-1 with an FFA titer of 8.5 g L-1. CONCLUSION: We showed that long-chain FFAs can be produced from metabolically engineered R. sphaeroides heterologously producing PLA2 in the periplasm. The FFA productivity was greatly increased by high-cell-density culture in two-phase culture with dodecane. This approach provides highly competitive productivity of long-chain FFAs by R. sphaeroides compared with other bacteria. This method may be applied to FFA production by other photosynthetic bacteria with similar differentiated membrane systems.

Biochemical characterization of protoporphyrinogen dehydrogenase and protoporphyrin ferrochelatase of Vibrio vulnificus and the critical complex formation between these enzymes.

BACKGROUND: Protoporphyrin IX (PPn), an intermediate in the heme biosynthesis reaction, generates singlet oxygen upon exposure to UV light. It has been proposed that PPn is channeled directly to ferrochelatase within a protoporphyrinogen dehydrogenase (PgdH1)-protoporphyrin ferrochelatase (PpfC) complex as a way to avoid this damaging side reaction. However, the PgdH1-PpfC complex has not been characterized, and the question of how heme affects the activities of PgdH1 has not been addressed. METHODS: Protein interactions were explored through pull-down assays and western blotting, and the importance of this complex in vivo was examined using inter-species combinations of the two proteins. The purified PgdH1-PpfC complex was characterized kinetically and used for heme binding studies. RESULTS: In Vibrio vulnificus, PgdH1 and PpfC formed an 8:8 heterohexadecameric complex that was important for maintaining PPn at low levels. PpfC catalyzed PPn efficiently whether or not it was part of the complex. Notably, heme was a noncompetitive inhibitor of V. vulnificus PgdH1, but a competitive inhibitor of the human protoporphyrinogen oxidase PgoX. CONCLUSION: The PdgH1-PpfC complex is important for protective channeling of PPn and for efficient catalysis of free PPn. The production of PPn by PgdH1 is regulated by feedback inhibition by heme. GENERAL SIGNIFICANCE: Both proteobacteria and eukaryotes have evolved mechanisms to prevent the harmful accumulation of the heme biosynthesis intermediate PPn. The data presented here suggest two previously unknown mechanisms: the channeling of PPn through the PgdH1-PpfC complex, and the direct inhibition of PgdH1 activity (PgoX activity as well) by heme.

The usefulness of low-dose CT scan in elderly patients with suspected acute lower respiratory infection in the emergency room.

OBJECTIVE: To evaluate the usefulness of low-dose CT (LDCT) for the diagnosis of acute lower respiratory infection (ALRI) in elderly patients in the emergency room (ER). METHODS: A total of 160 consecutive patients (mean age: 75.9 ± 9.2 years; range: 60-97 years), who were diagnosed to have ALRI by LDCT in the ER, were enrolled in this study. Initial chest radiograph (CR) and CT patterns of ALRI were analysed, and clinical courses of patients were assessed. RESULTS: 49 patients showed negative CR, in whom the main CT patterns were diffuse bronchial wall thickening (n = 23), ground-glass opacity (n = 6), mixed centrilobular nodules and ground-glass opacity (n = 3), small consolidation (n = 8) or consolidation in the dependent lung (n = 9), while the other 111 patients with the main CT pattern of consolidation demonstrated pulmonary abnormality on CR. Pulmonary oedema (12.5%) and pleural effusion (23.1%) were associated. The rate of hospitalization, care in the intensive care unit, mortality and comorbidity were significantly higher in the CR(+)LDCT(+) group (88.3%, 36.1%, 18.2% and 59.5%) than in the CR(-)LDCT(+) group (55.1%, 8.2%, 2.0% and 38.8%; p ≤ 0.05). CONCLUSION: LDCT was useful for the early diagnosis of ALRI in elderly patients who showed negative initial CR. The patients with negative initial CR had main CT patterns of diffuse bronchial wall thickening, ground-glass opacity, centrilobular nodules, small consolidation or consolidation in the dependent lung on LDCT. ADVANCES IN KNOWLEDGE: The use of LDCT may be considered for the early diagnosis of ALRI in elderly patients who have high comorbidity.

The Photoheterotrophic Growth of Bacteriochlorophyll Synthase-Deficient Mutant of Rhodobacter sphaeroides Is Restored by I44F Mutant Chlorophyll Synthase of Synechocystis sp. PCC 6803.

Chlorophyll synthase (ChlG) and bacteriochlorophyll synthase (BchG) have a high degree of substrate specificity. The BchG mutant of Rhodobacter sphaeroides, BG1 strain, is photosynthetically incompetent. When BG1 harboring chlG of Synechocystis sp. PCC 6803 was cultured photoheterotrophically, colonies arose at a frequency of approximately 10(-8). All the suppressor mutants were determined to have the same mutational change, ChlGI44F. The mutated enzyme ChlGI44F showed BchG activity. Remarkably, BchGF28I, which has the substitution of F at the corresponding 28(th) residue to I, showed ChlG activity. The Km values of ChlGI44F and BchGF28I for their original substrates, chlorophyllide (Chlide) a and bacteriochlorophyllide (Bchlide) a, respectively, were not affected by the mutations, but the Km values of ChlGI44F and BchGF28I for the new substrates Bchlide a and Chlide a, respectively, were more than 10-fold larger than those for their original substrates, suggesting the lower affinities for new substrates. Taken together, I44 and F28 are important for the substrate specificities of ChlG and BchG, respectively. The BchG activity of ChlGI44F and the ChlG activity of BchGF28I further suggest that ChlG and BchG are evolutionarily related enzymes.

VvpM induces human cell death via multifarious modes including necroptosis and autophagy.

VvpM, one of the extracellular metalloproteases produced by Vibrio vulnificus, induces apoptotic cell death via a pathway consisting of ERK activation, cytochrome c release, and activation of caspases-9 and -3. VvpM-treated cells also showed necrotic cell death as stained by propidium iodide (PI). The percentage of PI-stained cells was decreased by pretreatment with Necrostatin-1, indicating that VvpM-mediated cell death occurs through necroptosis. The appearance of autophagic vesicles and lipidated form of light-chain-3B in rVvpM-treated cells suggests an involvement of autophagy in this process. Therefore, the multifarious action of VvpM might be one of the factors responsible for V. vulnificus pathogenesis.

Role of HemF and HemN in the heme biosynthesis of Vibrio vulnificus under S-adenosylmethionine-limiting conditions.

Vibrio vulnificus contains two coproporphyrinogen III oxidases (CPOs): O2-dependent HemF and O2-independent HemN. The growth of the hemF mutant HF1 was similar to wild-type cells at pH 7.5 under 2% O2 conditions where HemN was active and had a half-life of 64 min. However, HF1 did not grow when the medium pH decreased to pH 5.0, where oxidative stress affects endogenous S-adenosylmethionine (SAM) levels. The growth of HF1 was restored not only by elevating the expression of MnSOD but also through the exogenous addition of SAM. For HF1 to grow under these SAM-limiting conditions, a mutation arose in hemN, encoding HemNY74F . Refolding of the denatured enzymes in vitro revealed that the apparent binding affinity of HemNY74F for the cofactor SAM1, which coordinates the 4Fe-4S cluster, was approximately sixfold higher than that of HemN. The Km of HemNY74F for the co-substrate SAM2, which provides radicals for CPO reactions, was threefold lower than that of HemN. Thus, affinities for both SAM1 and SAM2 were higher with the Y74F mutation. Taken together, when SAM is limiting, HemN is apparently nonfunctional, and heme synthesis is continued by HemF.

Effect of changes in the composition of cellular fatty acids on membrane fluidity of Rhodobacter sphaeroides.

The cellular fatty acid composition is important for metabolic plasticity in Rhodobacter sphaeroides. We explored the effects of changing the cellular ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs) in R. sphaeroides by overexpressing several key fatty acid biosynthetic enzymes through the use of expression plasmid pRK415. Bacteria containing the plasmid pRKfabI1 with the fabI1 gene that encodes enoyl-acyl carrier protein (ACP) reductase showed a reduction in the cellular UFA to SFA ratio from 4 (80% UFA) to 2 (65% UFA) and had decreased membrane fluidity and reduced cell growth. Additionally, the ratio of UFA to SFA of the chromatophore vesicles from pRKfabI1 -containing cells was similarly lowered, and the cell had decreased levels of light-harvesting complexes, but no change in intracytoplasmic membrane (ICM) content or photosynthetic (PS) gene expression. Both inhibition of enoyl- ACP reductase with diazaborine and addition of exogenous UFA restored membrane fluidity, cell growth, and the UFA to SFA ratio to wild-type levels in this strain. R. sphaeroides containing the pRKfabB plasmid with the fabB gene that encodes the enzyme ß-ketoacyl-ACP synthase I exhibited an increased UFA to SFA ratio from 4 (80% UFA) to 9 (90% UFA), but showed no change in membrane fluidity or growth rate relative to control cells. Thus, membrane fluidity in R. sphaeroides remains fairly unchanged when membrane UFA levels are between 80% and 90%, whereas membrane fluidity, cell growth, and cellular composition are affected when UFA levels are below 80%.

Enhanced photo-fermentative H2 production using Rhodobacter sphaeroides by ethanol addition and analysis of soluble microbial products.

BACKGROUND: Biological fermentation routes can provide an environmentally friendly way of producing H2 since they use renewable biomass as feedstock and proceed under ambient temperature and pressure. In particular, photo-fermentation has superior properties in terms of achieving high H2 yield through complete degradation of substrates. However, long-term H2 production data with stable performance is limited, and this data is essential for practical applications. In the present work, continuous photo-fermentative H2 production from lactate was attempted using the purple non-sulfur bacterium, Rhodobacter sphaeroides KD131. As a gradual drop in H2 production was observed, we attempted to add ethanol (0.2% v/v) to the medium. RESULTS: As continuous operation went on, H2 production was not sustained and showed a negligible H2 yield (< 0.5 mol H2/mol lactateadded) within two weeks. Electron balance analysis showed that the reason for the gradual drop in H2 production was ascribed to the increase in production of soluble microbial products (SMPs). To see the possible effect of ethanol addition, a batch test was first conducted. The presence of ethanol significantly increased the H2 yield from 1.15 to 2.20 mol H2/mol lactateadded, by suppressing the production of SMPs. The analysis of SMPs by size exclusion chromatography showed that, in the later period of fermentation, more than half of the low molecular weight SMPs (< 1 kDa) were consumed and used for H2 production when ethanol had been added, while the concentration of SMPs continuously increased in the absence of ethanol. It was found that the addition of ethanol facilitated the utilization of reducing power, resulting in an increase in the cellular levels of NAD(+) and NADP(+). In continuous operation, ethanol addition was effective, such that stable H2 production was attained with an H2 yield of 2.5 mol H2/mol lactateadded. Less than 15% of substrate electrons were used for SMP production, whereas 35% were used in the control. CONCLUSIONS: We have found that SMPs are the key factor in photo-fermentative H2 production, and their production can be suppressed by ethanol addition. However, since external addition of ethanol to the medium represents an extra economic burden, ethanol should be prepared in a cost-effective way.

Peroxidase and photoprotective activities of magnesium protoporphyrin IX.

Magnesium-protoporphyrin IX (Mg-PPn), which is formed through chelation of protoporphyrin IX (PPn) with Mg ion by Mg chelatase, is the first intermediate for the (bacterio)chlorophyll biosynthetic pathway. Interestingly, Mg-PPn provides peroxidase activity (approximately 4 × 10(-2) units/micrometer) detoxifying H2O2 in the presence of electron donor(s). The peroxidase activity was not detected unless PPn was chelated with Mg ion. Mg-PPn was found freely diffusible through the membrane of Escherichia coli and Vibrio vulnificus, protecting the cells from H2O2. Furthermore, unlike photosensitizers such as tetracycline and PPn, Mg-PPn did not show any phototoxicity, but rather it protected cell from ultraviolet (UV)-A-induced stress. Thus, the exogenous Mg-PPn could be used as an antioxidant and a UV block to protect cells from H2O2 stress and UV-induced damage.

MRI detection of intratumoral fat in hepatocellular carcinoma: potential biomarker for a more favorable prognosis.

OBJECTIVE: The purpose of this study was to determine whether the presence of intratumoral fat in hepatocellular carcinoma (HCC) could serve as an imaging biomarker to predict a favorable prognosis. MATERIALS AND METHODS: After a search of the radiology and pathology databases from January 2002 to December 2010, a cohort of patients with fat-containing HCC imaged by chemical-shift MRI techniques was matched with a cohort of patients with nonfat-containing HCC for TNM stage and type of subsequent treatment. The number and type of tumor progression, time to tumor progression (TTP), and overall survival (OS) were determined for each cohort. RESULTS: There were 46 patients included in each cohort. Tumor progression was more prevalent in the non-fat-containing HCC cohort (30 patients, 65.2%) compared with the fat-containing HCC cohort (16 patients, 34.7%; p = 0.001). Distant metastasis occurred more commonly in the non-fat-containing HCC cohort (10 patient, 21.7%) compared with the fat-containing HCC cohort (two patients, 4.3%; p = 0.039). The median TTP was significantly longer in the fat-containing HCC group (52 months) compared with the non-fat-containing HCC group (27 months; p = 0.037). The significantly longer TTP was primarily observed in the locoregional treatment subgroup (p = 0.028). No statistical significance in OS and subanalysis by treatment was observed (p = 0.63-0.81). CONCLUSION: Fat-containing HCC, imaged on an MRI unit, may predict a more favorable prognosis compared with nonfat-containing HCC.

Effect of carbon and nitrogen sources on photo-fermentative H2 production associated with nitrogenase, uptake hydrogenase activity, and PHB accumulation in Rhodobacter sphaeroides KD131.

During photo-fermentative H(2) production, the effects of carbon and nitrogen sources on nitrogenase and hydrogenase activity, poly-ß-hydroxybutyrate accumulation were investigated. In succinate/ammonium sulfate medium, H(2) was not detected for the first 6h because high ammonium concentration considerably reduced the nitrogenase activity to below 5 nmol/g-dcw/h. After 24h, 99% of the ammonium was consumed, and the nitrogenase activity increased to 296 nmol/g-dcw/h, accelerating H(2) production. In contrast, the ammonium in succinate/glutamate medium was much less, which led to rapid H(2) production in the beginning. However, H(2) evolution was repressed over time by increased ammonium. In the presence of H(2), hydrogenase activity increased with time regardless of the nitrogen source, and consequently, H(2) production was reduced. Compared with succinate, H(2) production in acetate media was severely limited due to increased pH over 9. During extended cultivation, the PHB accumulated in acetate media was 7 times higher than in succinate media.

Network identification and flux quantification of glucose metabolism in Rhodobacter sphaeroides under photoheterotrophic H(2)-producing conditions.

The nonsulfur purple bacteria that exhibit unusual metabolic versatility can produce hydrogen gas (H(2)) using the electrons derived from metabolism of organic compounds during photoheterotrophic growth. Here, based on (13)C tracer experiments, we identified the network of glucose metabolism and quantified intracellular carbon fluxes in Rhodobacter sphaeroides KD131 grown under H(2)-producing conditions. Moreover, we investigated how the intracellular fluxes in R. sphaeroides responded to knockout mutations in hydrogenase and poly-ß-hydroxybutyrate synthase genes, which led to increased H(2) yield. The relative contribution of the Entner-Doudoroff pathway and Calvin-Benson-Bassham cycle to glucose metabolism differed significantly in hydrogenase-deficient mutants, and this flux change contributed to the increased formation of the redox equivalent NADH. Disruption of hydrogenase and poly-ß-hydroxybutyrate synthase resulted in a significantly increased flux through the phosphoenolpyruvate carboxykinase and a reduced flux through the malic enzyme. A remarkable increase in the flux through the tricarboxylic acid cycle, a major NADH producer, was observed for the mutant strains. The in vivo regulation of the tricarboxylic acid cycle flux in photoheterotrophic R. sphaeroides was discussed based on the measurements of in vitro enzyme activities and intracellular concentrations of NADH and NAD(+). Overall, our results provide quantitative insights into how photoheterotrophic cells manipulate the metabolic network and redistribute intracellular fluxes to generate more electrons for increased H(2) production.

Complete genome sequence of Vibrio vulnificus MO6-24/O.

Vibrio vulnificus is the causative agent of life-threatening septicemia and severe wound infections. Here, we announce the complete annotated genome sequence of V. vulnificus MO6-24/O, isolated from a patient with septicemia. When it is compared with previously known V. vulnificus genomes, the genome of this bacterium shows a unique genetic makeup, including phagelike elements, carbohydrate metabolism-related genes, and the superintegron.

Silicon nanoparticles-graphene paper composites for Li ion battery anodes.

Composites of Si nanoparticles highly dispersed between graphene sheets, and supported by a 3-D network of graphite formed by reconstituting regions of graphene stacks exhibit high Li ion storage capacities and cycling stability. An electrode was prepared with a storage capacity >2200 mA h g(-1) after 50 cycles and >1500 mA h g(-1) after 200 cycles that decreased by <0.5% per cycle.

Competitive inhibitions of the chlorophyll synthase of Synechocystis sp. strain PCC 6803 by bacteriochlorophyllide a and the bacteriochlorophyll synthase of Rhodobacter sphaeroides by chlorophyllide a.

The photosynthetic growth of Synechocystis sp. strain PCC 6803 is hampered by exogenously added bacteriochlorophyllide a (Bchlide a) in a dose-dependent manner. The growth inhibition caused by Bchlide a, however, is relieved by an increased level of exogenously added chlorophyllide a (Chlide a). The results are explained by the competitive inhibition of chlorophyll synthase by Bchlide a, with inhibition constants (K(I)s) of 0.3 mM and 1.14 mM in the presence of sufficient geranylgeranyl pyrophosphate (GGPP) and phytyl pyrophosphate (PPP), respectively. Surprisingly, the bacteriochlorophyll synthase of Rhodobacter sphaeroides is inhibited competitively by Chlide a, with K(I)s of 0.54 mM and 0.77 mM in the presence of sufficient GGPP and PPP, respectively. Consistently, exogenously added Chlide a inhibits the metabolic conversion of exogenously added Bchlide a to bacteriochlorophyll a by an R. sphaeroides bchFNB-bchZ mutant that neither synthesizes nor metabolizes Chlide a. The metabolic inhibition by Chlide a, however, is relieved by the elevated level of Bchlide a. Thus, the chlorophyll synthase of Synechocystis sp. PCC 6803 and the bacteriochlorophyll synthase of R. sphaeroides, both of which perform ping-pong-type reactions, are inhibited by Bchlide a and Chlide a, respectively. Although neither inhibitor is catalyzed by the target enzyme, inhibitions in the competitive mode suggest a structural similarity between their active sites.

Cadaverine is transported into Vibrio vulnificus through its CadB in alkaline environment.

The exogenously-added cadaverine is effective in protecting Vibrio vulnificus from methyl viologen (MV)-induced superoxide stress at pH 8.5. Such a protective effect by cadaverine was not observed at pH 7.5. Consistently, the accumulated level of intracellular cadaverine at pH 8.5 is approximately four times as much as that of the control cell at pH 7.5. Cadaverine accumulation is not affected by MV. The protection of V. vulnificus by cadaverine from superoxide stress was abolished when cadB coding for lysine-cadaverine antiporter was interrupted. However, the cadaverine-mediated protection was complemented with cadB DNA. Therefore, CadB of V. vulnificus not only acts as a lysine-cadaverine antiporter at acid pH to neutralize the external medium but also mediates cadaverine uptake at alkaline pH to result in cell protection from superoxide stress.