Jeong K. Lee

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.

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-β-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 H2-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-β-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-β-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 Rhodobacter sphaeroides KD131

Rhodobacter sphaeroides is a purple nonsulfur photosynthetic bacterium that is considered a possible source of H(2) production. R. sphaeroides KD131, which was isolated from sea mud in South Korea, was found to produce high levels of H(2). Here we report the complete and annotated genome sequence of R. sphaeroides KD131.

Growth arrest of Synechocystis sp. PCC6803 by superoxide generated from heterologously expressed Rhodobacter sphaeroides chlorophyllide a reductase

The photosynthetic growth of Synechocystis sp. PCC6803 ceased upon expression of Rhodobacter sphaeroides chlorophyllide a reductase (COR). However, an increase in cytosolic superoxide dismutase level in the recombinant Synechocystis sp. PCC6803 completely reversed the growth cessation. This demonstrates that COR generates superoxide in Synechocystis sp. PCC6803. Considering the dissolved oxygen (DO) level suitable for COR, the intracellular DO of this oxygenic photosynthetic cell appears to be low enough to support COR‐mediated superoxide generation. The growth arrest of Synechocystis sp. PCC6803 by COR may give an insight into the evolutionary path from bacteriochlorophyll a biosynthetic pathway to chlorophyll a, which bypasses COR reaction.

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.

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.

Characterization of ATPase Activity of Free and Immobilized Chromatophore Membrane Vesicles of Rhodobacter sphaeroides

The intracytoplasmic membrane of Rhodobacter sphaeroides readily vesiculates when cells are lysed. The resulting chromatophore membrane vesicle (CMV) contains the photosynthetic machineries to synthesize ATP by ATPase. The light-dependent ATPase activity of CMV was lowered in the presence of O₂, but the activity increased to the level observed under anaerobic condition when the reaction mixture was supplemented with ascorbic acid (≥0.5 mM). Cell lysis in the presence of biotinyl cap phospholipid (bcp) resulted in the incorporation of bcp into the membrane to form biotinylated CMV (bCMV), which binds to streptavidin resin at a ratio of approximately 24 μg bacteriochlorophyll a/ml resin. The ATPase activity of CMV was not affected by biotinylation, but approximately 30% of the activity was lost by immobilization to resin. Interestingly, the remaining 70% of ATPase activity stayed constant during 7-day storage at 4°C. On the contrary, the ATPase activity of bCMV without immobilization gradually decreased to approximately 40% of the initial level in the same comparison. Thus, the ATPase activity of CMV is sustainable after immobilization, and the immobilized bCMV can be used repeatedly as an ATP generator.