Enhancement of NAD(H) pool for formation of oxidized biochemicals in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The NAD⁺/NADH ratio and the total NAD(H) play important roles for whole-cell biochemical redox transformations. After the carbon source is exhausted, the degradation of NAD(H) could contribute to a decline in the rate of a desired conversion. In this study, methods to slow the native rate of NAD(H) degradation were examined using whole-cell Escherichia coli with two model oxidative NAD⁺-dependent biotransformations. A high phosphate concentration (50 mM) was observed to slow NAD(H) degradation. We also constructed E. coli strains with deletions in genes coding several enzymes involved in NAD⁺ degradation. In shake-flask experiments, the total NAD(H) concentration positively correlated with conversion of xylitol to l-xylulose by xylitol 4-dehydrogenase, and the greatest conversion (80%) was observed using MG1655 nadR nudC mazG/pZE12-xdh/pCS27-nox. Controlled 1-L batch processes comparing E. coli nadR nudC mazG with a wild-type background strain demonstrated a 30% increase in final l-xylulose concentration (5.6 vs. 7.9 g/L) and a 25% increase in conversion (0.53 vs. 0.66 g/g). MG1655 nadR nudC mazG was also examined for the conversion of galactitol to l-tagatose by galactitol 2-dehydrogenase. A batch process using 15 g/L glycerol and 10 g/L galactitol generated over 9.4 g/L l-tagatose, corresponding to 90% conversion and a yield of 0.95 g l-tagatose/g galactitol consumed. The results demonstrate the value of minimizing NAD(H) degradation as a means to improve NAD⁺-dependent biotransformations.     

<Emphasis Type="Italic">Halomonas tibetensis</Emphasis> sp. nov., isolated from saline lakes on Tibetan Plateau

Strains pyc13^T and ZGT13 were isolated from Lake Pengyan and Lake Zigetang on Tibetan Plateau, respectively. Both strains were Gram-negative, catalase- and oxidase-positive, aerobic, rod-shaped, nonmotile, and nonflagellated bacteria. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains pyc13^T and ZGT13 belong to the genus Halomonas, with Halomonas alkalicola 56-L4-10aEn^T as their closest neighbor, showing 97.4% 16S rRNA gene sequence similarity. The predominant respiratory quinone of both strains was Q-9, with Q-8 as a minor component. The major fatty acids of both strains were C_18:1ω6c/C_18:1ω7c, C_16:1ω6c/C_16:1ω7c, C_16:0, and C_12:0 3OH. The polar lipids of both strains consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, glycolipid, phospholipids of unknown structure containing glucosamine, and unidentified phospholipids. The DNA G + C content of pyc13^T and ZGT13 were 62.6 and 63.4 mol%, respectively. The DNA-DNA hybridization values of strain pyc13^T were 34, 41, 61, 35, and 35% with the reference strains H. alkalicola 56-L4-10aEn^T, H. sediminicola CPS11^T, H. mongoliensis Z-7009^T, H. ventosae Al12^T, and H. fontilapidosi 5CR^T, respectively. Phenotypic, biochemical, genotypic, and DNA-DNA hybridization data showed that strains pyc13^T and ZGT13 represent a new species within the genus Halomonas, for which the name H. tibetensis sp. nov. is proposed. The type strain is pyc13^T (= CGMCC 1.15949^T = KCTC 52660^T).     

The Structure of Calf Liver Cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript> at 2.8 Å Resolution

CYTOCHROME b₅ is a haem-containing protein in the microsomes of liver tissue. It interacts specifically with a flavo-protein, cytochrome b₅ reductase, which catalyses the transfer of electrons from NADH to the haem iron of the cytochrome¹. The microsomal cytochrome b₅ system has been implicated in fatty acid desaturation reactions² and a similar system in erythrocytes may catalyse the reduction of methaemoglobin³. Calf liver cytochrome b₅, solubilized by pancreatic lipase, has a molecular weight of 11,000 and consists of ninety-three amino-acids in the sequence shown in Fig. 1 (refs. 4 and 5). The haem group is non-covalently bound to the protein and can be removed reversibly by acid acetone treatment⁶.     

<Emphasis Type="Italic">Arcobacter acticola</Emphasis> sp. nov., isolated from seawater on the East Sea in South Korea

A Gram-stain-negative, facultative aerobic, non-flagellated, and rod-shaped bacterium, designated AR-13^T, was isolated from a seawater on the East Sea in South Korea, and subjected to a polyphasic taxonomic study. Strain AR-13^T grew optimally at 30°C, at pH 7.0–8.0 and in the presence of 0–0.5% (w/v) NaCl. The phylogenetic trees based on 16S rRNA gene sequences showed that strain AR-13^T fell within the clade comprising the type strains of Arcobacter species, clustering coherently with the type strain of Arcobacter venerupis. Strain AR-13^T exhibited 16S rRNA gene sequence similarity values of 98.1% to the type strain of A. venerupis and of 93.2–96.9% to the type strains of the other Arcobacter species. Strain AR-13^T contained MK-6 as the only menaquinone and summed feature 3 (C_16:1ω7c and/or C_16:1ω6c), C_16:0, C_18:1ω7c, and summed feature 2 (iso-C_16:1 I and/or C_14:0 3-OH) as the major fatty acids. The major polar lipids detected in strain AR-13^T were phosphatidylethanolamine, phosphatidylglycerol, and one unidentified aminophospholipid. The DNA G+C content was 28.3 mol% and its mean DNA-DNA relatedness value with the type strain of A. venerupis was 21%. Differential phenotypic properties, together with its phylogenetic and genetic distinctiveness, revealed that strain AR-13^T is separated from recognized Arcobacter species. On the basis of the data presented, strain AR-13^T is considered to represent a novel species of the genus Arcobacter, for which the name Arcobacter acticola sp. nov. is proposed. The type strain is AR-13^T (=KCTC 52212^T =NBRC 112272^T).     

Screening and identification of an <Emphasis Type="Italic">Enterobacter ludwigii</Emphasis> strain expressing an active <Emphasis Type="Italic">β</Emphasis>-xylosidase

Researchers have expressed increasing interest in the xylanolytic enzymes used in hemicellulose hydrolysis that convert wood and agricultural residues to second-generation biofuels. In our study, 32 isolates showed clear hydrolysis zones on agar plates containing xylan after Congo red staining. Among these isolates, strain LY-62 exhibited the highest β-xylosidase activity (1.29?±?0.05 U/mL). According to the phylogenetic analysis of the 16S rDNA, strain LY-62 belongs to the Enterobacter genus. Using a combination of electron microscopy, Gram-staining, and conventional physiological and biochemical examinations, the strain LY-62 was identified as Enterobacter ludwigii. The β-xylosidase gene from Enterobacter ludwigii LY-62 was cloned, and the full-length protein was expressed in Escherichia coli as an N-terminal or C-terminal His-tagged fusions protein. Optimal β-xylosidase activity was achieved at pH 7.0 and 40 °C. The Michaelis constant K_M values for His-Xyl62 and Xyl62-His were 1.55 and 2.8 mmol/L, respectively. The k_cat values for His-Xyl62 and Xyl62-His were 8.51 and 6.94 s^?1, respectively. The catalytic efficiencies of His-Xyl62 and Xyl62-His were 5.49 and 2.48 s^?1?×?mM^?1, respectively. Thus, Xyl62 is a functional β-xylosidase, and our study represents the first report of a β-xylosidase from Enterobacter ludwigii.     

Characterization of alcohol dehydrogenase from <Emphasis Type="Italic">Kangiella koreensis</Emphasis> and its application to production of all-<Emphasis Type="Italic">trans</Emphasis>-retinol

A recombinant alcohol dehydrogenase (ADH) from Kangiella koreensis was purified as a 40 kDa dimer with a specific activity of 21.3 nmol min^?1 mg^?1, a K _m of 1.8 μM, and a k _cat of 1.7 min^?1 for all-trans-retinal using NADH as cofactor. The enzyme showed activity for all-trans-retinol using NAD ⁺ as a cofactor. The reaction conditions for all-trans-retinol production were optimal at pH 6.5 and 60 °C, 2 g enzyme l^?1, and 2,200 mg all-trans-retinal l^?1 in the presence of 5 % (v/v) methanol, 1 % (w/v) hydroquinone, and 10 mM NADH. Under optimized conditions, the ADH produced 600 mg all-trans-retinol l^?1 after 3 h, with a conversion yield of 27.3 % (w/w) and a productivity of 200 mg l^?1 h^?1. This is the first report of the characterization of a bacterial ADH for all-trans-retinal and the biotechnological production of all-trans-retinol using ADH.     

Improvement of 1,3-propanediol oxidoreductase (DhaT) stability against 3-hydroxypropionaldehyde by substitution of cysteine residues

1,3-propanediol oxidoreductase (DhaT), which catalyzes the conversion of 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) with the oxidation of NADH to NAD⁺, is a key enzyme in the production of 1,3-PD from glycerol. DhaT is known to be severely inactivated by its physiological substrate, 3-HPA, due to the reaction of 3-HPA with the thiol group of the cysteine residues. In this study, using site-directed mutagenesis, four cysteine residues in Klebsiella pneumoniae J2B DhaT were substituted to alanine, the amino acid commonly found in cysteine’s positions in other DhaT, individually and in combination. Among the total of 15 mutants developed, a double mutant (C28A_C107A) and a triple mutant (C28A_C93A_C107A) exhibited approximately 50 and 16% higher activity than the wild-type counterpart, respectively, after 1 h incubation with 10 mM 3-HPA. According to detailed kinetic studies, the double mutant had slightly better kinetic properties (V _max , K _cat , and K _m for both 3-HPA and NADH) than wild-type DhaT. This study shows that DhaT stability against 3-HPA can be increased by cysteine-residue removal, albeit to a limited extent.     

Reassessment of hydrogen tolerance in <Emphasis Type="Italic">Caldicellulosiruptor saccharolyticus</Emphasis>

Background

Caldicellulosiruptor saccharolyticus has the ability to produce hydrogen (H₂) at high yields from a wide spectrum of carbon sources, and has therefore gained industrial interest. For a cost-effective biohydrogen process, the ability of an organism to tolerate high partial pressures of H₂ (P_H2) is a critical aspect to eliminate the need for continuous stripping of the produced H₂ from the bioreactor.

Results

Herein, we demonstrate that, under given conditions, growth and H₂ production in C. saccharolyticus can be sustained at P_H2 up to 67 kPa in a chemostat. At this P_H2, 38% and 16% of the pyruvate flux was redirected to lactate and ethanol, respectively, to maintain a relatively low cytosolic NADH/NAD ratio (0.12 mol/mol). To investigate the effect of the redox ratio on the glycolytic flux, a kinetic model describing the activity of the key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was developed. Indeed, at NADH/NAD ratios of 0.12 mol/mol (K i of NADH = 0.03 ± 0.01 mM) GAPDH activity was inhibited by only 50% allowing still a high glycolytic flux (3.2 ± 0.4 mM/h). Even at high NADH/NAD ratios up to 1 mol/mol the enzyme was not completely inhibited. During batch cultivations, hydrogen tolerance of C. saccharolyticus was dependent on the growth phase of the organism as well as the carbon and energy source used. The obtained results were analyzed, based on thermodynamic and enzyme kinetic considerations, to gain insight in the mechanism underlying the unique ability of C. saccharolyticus to grow and produce H₂ under relatively high P_H2.

Conclusion

C. saccharolyticus is able to grow and produce hydrogen at high P_H2, hence eliminating the need of gas sparging in its cultures. Under this condition, it has a unique ability to fine tune its metabolism by maintaining the glycolytic flux through regulating GAPDH activity and redistribution of pyruvate flux. Concerning the later, xylose-rich feedstock should be preferred over the sucrose-rich one for better H₂ yield.

     

Analysis of applicability of triplet-state emission of molecular hydrogen for spectral diagnostics of a DC discharge

The applicability of emission of the N ³Λ_σ triplet states of molecular hydrogen for spectral diagnostics of the positive column of a dc glow discharge in hydrogen at translational gas temperatures of 360–600 K, specific absorbed powers of 0.8–4.25 W/cm, gas pressures of p = 0.3–15.0 Torr, reduced fields of E/N = 30–130 Td, and electron densities of n _e = 4.0 × 10⁹–6.5 × 10¹⁰ cm^–3 is analyzed by using an advanced level-based semi-empirical collisional?radiative model. It is found that secondary processes make the main contribution to the population and decay of the N ³Λ_σ = a ³Σ⁺ _g , c ³Π _u , g ³Σ⁺ _g , h ³Σ⁺ _g , and i ³Π _g triplet states. The dipole-allowed transitions e ³Σ⁺ _g → a ³Σ⁺ _g , f ³Σ⁺ _g → a ³Σ⁺ _g , g ³Σ⁺ _g and k ³Π _u → a ³Σ⁺ _g can be used for spectral diagnostics of a dc discharge within a simplified coronal model.     

A <Emphasis Type="Italic">bean common mosaic virus</Emphasis> (BCMV)-resistance gene is fine-mapped to the same region as <Emphasis Type="Italic">Rsv1</Emphasis>-<Emphasis Type="Italic">h</Emphasis> in the soybean cultivar Suweon 97

Key message

In the soybean cultivar Suweon 97, BCMV-resistance gene was fine-mapped to a 58.1-kb region co-localizing with the Soybean mosaic virus (SMV)-resistance gene, Rsv1-h raising a possibility that the same gene is utilized against both viral pathogens.

Abstract

Certain soybean cultivars exhibit resistance against soybean mosaic virus (SMV) or bean common mosaic virus (BCMV). Although several SMV-resistance loci have been reported, the understanding of the mechanism underlying BCMV resistance in soybean is limited. Here, by crossing a resistant cultivar Suweon 97 with a susceptible cultivar Williams 82 and inoculating 220 F₂ individuals with a BCMV strain (HZZB011), we observed a 3:1 (resistant/susceptible) segregation ratio, suggesting that Suweon 97 possesses a single dominant resistance gene against BCMV. By performing bulked segregant analysis with 186 polymorphic simple sequence repeat (SSR) markers across the genome, the resistance gene was determined to be linked with marker BARSOYSSR_13_1109. Examining the genotypes of nearby SSR markers on all 220 F₂ individuals then narrowed down the gene between markers BARSOYSSR_13_1109 and BARSOYSSR_13_1122. Furthermore, 14 previously established F_2:3 lines showing crossovers between the two markers were assayed for their phenotypes upon BCMV inoculation. By developing six more SNP (single nucleotide polymorphism) markers, the resistance gene was finally delimited to a 58.1-kb interval flanked by BARSOYSSR_13_1114 and SNP-49. Five genes were annotated in this interval of the Williams 82 genome, including a characteristic coiled-coil nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR, CNL)-type of resistance gene, Glyma13g184800. Coincidentally, the SMV-resistance allele Rsv1-h was previously mapped to almost the same region, thereby suggesting that soybean Suweon 97 likely relies on the same CNL-type R gene to resist both viral pathogens.

     

The Peptide-Mediated Interactions Between Human Osteoclast-Stimulating Factor and Its Partner Proteins in Osteoporosis: Which Binds to Which?

Human osteoclast-stimulating factor (hOSF) is an intracellular protein produced by osteoclasts that induces osteoclast formation and bone resorption in osteoporosis by recruiting multiple signaling complexes with its diverse biological partners through peptide-mediated interactions (PMIs). The protein contains a modular peptide-recognition domain of Src homology 3 (SH3), which can recognize and bind to the polyproline regions of its partner proteins, as well as two N-terminal polyproline segments, which can be recognized and bound by the SH3 domains of its partner proteins. Here, we attempted to elucidate the complicated PMIs between the different SH3 domains and different polyprolines of hOSF and its three known interacting partners, i.e. proto-oncogene tyrosine-protein kinase (c-Src), survival motor neuron (SMN) and Src-associated in mitosis, 68 kD (Sam68). A total of 29 peptide segments containing the SH3-binding motif PXXP were extracted from these partner proteins, which are potential binding sites of hOSF SH3 domain, while the c-Src kinase also possesses a SH3 domain that may recognize and bind the two polyproline peptides at hOSF N-terminus. Structural bioinformatics analysis identified a number of biologically functional PMI candidates between these SH3 domains and these polyproline peptides, which were then tested in vitro using fluorescence spectroscopy assays. Consequently, it is found that (i) hOSF SH3 domain exhibits strong binding potency to two Sam68 peptides ³⁶RQPPLPHR⁴³ (K _d = 13.7 μM) and ⁴²⁵APPARPVK⁴³² (K _d = 3.2 μM) as well as moderate affinity to three SMN peptides ¹⁹³FLPPPPPM²⁰⁰ (K _d = 56.2 μM), ²³⁵PFPSGPPI²⁴² (K _d = 28.4 μM) and ²⁴⁶PPPICPDS²⁵³ (K _d = 74.5 μM), but has only weak or no binding to c-Src peptides. Instead, a proline-rich region at hOSF N-terminal that contains two overlapping peptides (³KPPPKPVK¹⁰ and ⁶PKPVKPGQ¹³) can be bound tightly by c-Src SH3 domain with high and moderate affinity (K _d = 5.8 and 39.6 μM, respectively).     

Assessment of photosynthetic potential of indoor plants under cold stress

Photosynthetic parameters including net photosynthetic rate (P_N), transpiration rate (E), water-use efficiency (WUE), and stomatal conductance (g_s) were studied in indoor C₃ plants Philodendron domesticum (Pd), Dracaena fragans (Df), Peperomia obtussifolia (Po), Chlorophytum comosum (Cc), and in a CAM plant, Sansevieria trifasciata (St), exposed to various low temperatures (0, 5, 10, 15, 20, and 25°C). All studied plants survived up to 0°C, but only St and Cc endured, while other plants wilted, when the temperature increased back to room temperature (25°C). The P_N declined rapidly with the decrease of temperature in all studied plants. St showed the maximum P_N of 11.9 μmol m^?2 s^?1 at 25°C followed by Cc, Po, Pd, and Df. E also followed a trend almost similar to that of P_N. St showed minimum E (0.1 mmol m^?2 s^?1) as compared to other studied C₃ plants at 25°C. The E decreased up to ≈4-fold at 5 and 0°C. Furthermore, a considerable decline in WUE was observed under cold stress in all C₃ plants, while St showed maximum WUE. Similarly, the g_s also declined gradually with the decrease in the temperature in all plants. Among C₃ plants, Pd and Po showed the maximum g_s of 0.07 mol m^?2 s^?1 at 25°C followed by Df and Cc. However, St showed the minimum g_s that further decreased up to ～4-fold at 0°C. In addition, the content of photosynthetic pigments [chlorophyll a, b, (a+b), and carotenoids] was varying in all studied plants at 0°C. Our findings clearly indicated the best photosynthetic potential of St compared to other studied plants. This species might be recommended for improving air quality in high-altitude closed environments.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Impact of two-bond <Superscript>15</Superscript>N–<Superscript>15</Superscript>N scalar couplings on <Superscript>15</Superscript>N transverse relaxation measurements for arginine side chains of proteins

NMR relaxation of arginine (Arg) ¹⁵N_ε nuclei is useful for studying side-chain dynamics of proteins. In this work, we studied the impact of two geminal ¹⁵N–¹⁵N scalar couplings on measurements of transverse relaxation rates (R ₂ ) for Arg side-chain ¹⁵N_ε nuclei. For 12 Arg side chains of the DNA-binding domain of the Antp protein, we measured the geminal ¹⁵N–¹⁵N couplings ( ² J _NN ) of the ¹⁵N_ε nuclei and found that the magnitudes of the ² J _NN coupling constants were virtually uniform with an average of 1.2 Hz. Our simulations, assuming ideal 180° rotations for all ¹⁵N nuclei, suggested that the two ² J _NN couplings of this magnitude could in principle cause significant modulation in signal intensities during the Carr–Purcell-Meiboom–Gill (CPMG) scheme for Arg ¹⁵N_ε R ₂ measurements. However, our experimental data show that the expected modulation via two ² J _NN couplings vanishes during the ¹⁵N CPMG scheme. This quenching of J modulation can be explained by the mechanism described in Dittmer and Bodenhausen (Chemphyschem 7:831–836, 2006). This effect allows for accurate measurements of R ₂ relaxation rates for Arg side-chain ¹⁵N_ε nuclei despite the presence of two ² J _NN couplings. Although the so-called recoupling conditions may cause overestimate of R ₂ rates for very mobile Arg side chains, such conditions can readily be avoided through appropriate experimental settings.     

Cytosolic monodehydroascorbate reductase gene affects stress adaptation and grain yield under paddy field conditions in <Emphasis Type="Italic">Oryza sativa L. japonica</Emphasis>

Monodehydroascorbate reductase (MDHAR), which is responsible for growth, development and stress response in plants, is a key enzyme in the maintenance of the ascorbate (AsA) pool through the AsA–glutathione (AsA–GSH) cycle and is induced by abiotic stresses. It has highly conserved regions containing FAD- and NAD(P)H-binding domains. In particular, NAD(P)H is a significant electron donor in the AsA–GSH pathway. In this context, we introduced RNA interference (RNAi) to determine the functional role of Oryza sativa L. japonica MDHAR isoform 3 (OsMDHAR3) and developed transgenic (mdhar3) rice plants in which the NAD(P)H domain was silenced. The mdhar3 rice plants were more sensitive to salt stress than the wild-type (WT) plants. In addition, the mdhar3 rice plants showed decreased ability for environmental adaptation because of an imbalance in the redox homeostasis and reduced AsA pool. These plants showed increased hydroperoxide levels and ion leakage, and decreased chlorophyll content and ascorbate/dehydroascorbate ratio under the paddy field conditions; they also exhibited a reduction in the total biomass and grain yield. Furthermore, the activity of a purified E196A mutant of the OsMDHAR protein decreased to approximately 70% of the activity of the WT protein. These results suggest that OsMDHAR3 plays a critical role in the intrinsic resistance, as well as in the sensitivity of seed maturation and productivity, of rice plants to environmental stresses, thereby indicating the functional importance of NADH in MDHAR activity, in vivo and in vitro.     

Microwave electrode discharge in nitrogen: Structure and characteristics of the electrode region

The parameters of the electrode region of an electrode microwave discharge in nitrogen are studied by emission spectroscopy. The radial and axial distributions of the intensities of the bands of the second (N₂(C ³Π _u → B ³Π _g )) and first (N₂(B ³Π _g → A ³Σ _u ⁺ )) positive systems of molecular nitrogen and the first negative system of nitrogen ions (N ₂ ⁺ (B ²Σ _u ⁺ → X ²Σ _g ⁺ )), the radial profiles of the electric field E and the electron density N _e , and the absolute populations of the vibrational levels v _C = 0–4 of the C ³Π _u excited state of N₂ and the vibrational level v _Bi = 0 of the B ²Σ _u ⁺ excited state of a molecular nitrogen ion are determined. The population temperature of the first vibrational level T _V of the ground electronic state X ¹Σ _g ⁺ of N₂ and the excitation temperature T _C of the C ³Π _u state in the electrode region of the discharge are measured. The radius of the spherical region and the spatially integrated plasma emission spectra are studied as functions of the incident microwave power and gas pressure. A method for determining the electron density and the microwave field strength from the plasma emission characteristics is described in detail.     

Natively oxidized amino acid residues in the spinach cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex

The cytochrome b ₆ f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b ₆ f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc₁ complex. The types of ROS produced (O₂^{??, 1}O₂, and, possibly, H₂O₂) and the site(s) of ROS production within the b ₆ f complex have been the subject of some debate. Proposed sources of ROS have included the heme b _p , PQ _p ^?? (possible sources for O₂^??), the Rieske iron–sulfur cluster (possible source of O₂^?? and/or ¹O₂), Chl a (possible source of ¹O₂), and heme c _n (possible source of O₂^?? and/or H₂O₂). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b ₆ f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b ₆ f complex.     

Antirestriction and antimodification activities of T7 Ocr: Effects of amino acid substitutions in the interface

The T7 antirestriction protein Ocr, encoded by 0.3 (ocr), specifically inhibits ATP-dependent type I restriction-modification systems. T7 0.3 (ocr) was cloned in pUC18. Ocr inhibited both restriction and modification activities of the type I restriction-modification system (EcoKI) in Escherichia coli K12. The Ocr F53D A57E mutant was obtained and proved to inhibit only restriction activity of EcoKI. The 0.3 (ocr) and Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P _ltetO-1 promoter, strongly controlled by the TetR repressor. The bioluminescence intensity and luciferase content varied up to 5000-fold in E. coli K12 MG1655Z1 tetR⁺ (pZE21-luxCDABE) cells, depending on the environmental concentration of the inductor anhydrotetracycline. The antirestriction activity of Ocr and Ocr F53D A57E was studied as a function of their concentration in the cell. The dissociation constant K _d, characterizing the binding with EcoKI, differed 1000-fold between Ocr and Ocr F53D A57E (10^?10 M versus 10^?7 M).