Phosphorylation and Functional Properties of the IIA Domain of the Lactose Transport Protein of Streptococcus thermophilus

The lactose-H⁺ symport protein (LacS) of Streptococcus thermophilus has a carboxyl-terminal regulatory domain (IIA^LacS) that is homologous to a family of proteins and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in various organisms, of which IIA^Glc of Escherichia coli is the best-characterized member. On the basis of these similarities, it was anticipated that IIA^LacS would be able to perform one or more functions associated with IIA^Glc, i.e., carry out phosphoryl transfer and/or affect other catabolic functions. The gene fragment encoding IIA^LacS was overexpressed in Escherichia coli, and the protein was purified in two steps by metal affinity and anion-exchange chromatography. IIA^LacS was unable to restore glucose uptake in a IIA^Glc-deficient strain, which is consistent with a very low rate of phosphorylation of IIA^LacS by phosphorylated HPr (HPr~P) from E. coli. With HPr~P from S. thermophilus, the rate was more than 10-fold higher, but the rate constants for the phosphorylation of IIA^LacS (k₁ = 4.3 × 10² M⁻¹ s⁻¹) and dephosphorylation of IIA^LacS~P by HPr (k₋₁ = 1.1 × 10³ M⁻¹ s⁻¹) are still at least 4 orders of magnitude lower than for the phosphoryltransfer between IIA^Glc and HPr from E. coli. This finding suggests that IIA^LacS has evolved into a protein domain whose main function is not to transfer phosphoryl groups rapidly. On the basis of sequence alignment of IIA proteins with and without putative phosphoryl transfer functions and the known structure of IIA^Glc, we constructed a double mutant [IIA^LacS(I548E/G556D)] that was predicted to have increased phosphoryl transfer activity. Indeed, the phosphorylation rate of IIA^LacS(I548E/G556D) by HPr~P increased (k₁ = 4.0 × 10³ M⁻¹ s⁻¹) and became nearly independent of the source of HPr~P (S. thermophilus, Bacillus subtilis, or E. coli). The increased phosphoryl transfer rate of IIA^LacS(I548E/G556D) was insufficient to complement IIA^Glc in PTS-mediated glucose transport in E. coli. Both IIA^LacS and IIA^LacS(I548E/G556D) could replace IIA^Glc, but in another function: they inhibited glycerol kinase (inducer exclusion) when present in the unphosphorylated form.     

Engineering of Acetate Recycling and Citrate Synthase to Improve Aerobic Succinate Production in Corynebacterium glutamicum

Corynebacterium glutamicum lacking the succinate dehydrogenase complex can produce succinate aerobically with acetate representing the major byproduct. Efforts to increase succinate production involved deletion of acetate formation pathways and overexpression of anaplerotic pathways, but acetate formation could not be completely eliminated. To address this issue, we constructed a pathway for recycling wasted carbon in succinate-producing C. glutamicum. The acetyl-CoA synthetase from Bacillus subtilis was heterologously introduced into C. glutamicum for the first time. The engineered strain ZX1 (pEacsA) did not secrete acetate and produced succinate with a yield of 0.50 mol (mol glucose)⁻¹. Moreover, in order to drive more carbon towards succinate biosynthesis, the native citrate synthase encoded by gltA was overexpressed, leading to strain ZX1 (pEacsAgltA), which showed a 22% increase in succinate yield and a 62% decrease in pyruvate yield compared to strain ZX1 (pEacsA). In fed-batch cultivations, strain ZX1 (pEacsAgltA) produced 241 mM succinate with an average volumetric productivity of 3.55 mM h⁻¹ and an average yield of 0.63 mol (mol glucose) ⁻¹, making it a promising platform for the aerobic production of succinate at large scale.     

Denitrification by Chromobacterium violaceum

One host (Rana catesbiana)-associated and two free-living mesophilic strains of bacteria with violet pigmentation and biochemical characteristics of Chromobacterium violaceum were isolated from freshwater habitats. Cells of each freshly isolated strain and of strain ATCC 12472 (the neotype strain) grew anaerobically with glucose as the sole carbon and energy source. The major fermentation products of cells grown in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) supplemented with glucose included acetate, small amounts of propionate, lactate, and pyruvate. The final cell yield and culture growth rate of each strain cultured anaerobically in this medium increased approximately twofold with the addition of 2 mM NaNO₃. Final growth yields increased in direct proportion to the quantity of added NaNO₃ over the range of 0.5 to 5 mM. Each strain reduced NO₃⁻, producing NO₂⁻, NO, and N₂O. NO₂⁻ accumulated transiently. With 2 mM NaNO₃ in the medium, N₂O made up 85 to 98% of the N product recovered with each strain. N-oxides were recovered in the same quantity and distribution whether 0.01 atm (ca. 1 kPa) of C₂H₂ (added to block N₂O reduction) was present or not. Neither N₂ production nor gas accumulation was detected during NO₃⁻ reduction by growing cells. Cell growth in media containing 0.5 to 5 mM NaNO₂ in lieu of NaNO₃ was delayed, and although N₂O was produced by the end of growth, NO₂⁻ -containing media did not support growth to an extent greater than did medium lacking NO₃⁻ or NO₂⁻. The data indicate that C. violaceum cells ferment glucose or denitrify, terminating denitrification with the production of N₂O, and that NO₂⁻ reduction to N₂O is not coupled to growth but may serve as a detoxification mechanism. No strain detectably fixed N₂ (reduced C₂H₂).     

Identification and Enzymatic Characterization of the Maltose-Inducible α-Glucosidase MalL (Sucrase-Isomaltase-Maltase) of Bacillus subtilis

A gene coding for a putative α-glucosidase has been identified in the open reading frame yvdL (now termed malL), which was sequenced as part of the Bacillus subtilis genome project. The enzyme was overproduced in Escherichia coli and purified. Further analyses indicate that MalL is a specific oligo-1,4-1,6-α-glucosidase (sucrase-maltase-isomaltase). MalL expression in B. subtilis requires maltose induction and is subject to carbon catabolite repression by glucose and fructose. Insertional mutagenesis of malL resulted in a complete inactivation of the maltose-inducible α-glucosidase activity in crude protein extracts and a Mal⁻ phenotype.     

Thermodynamics of Formate-Oxidizing Metabolism and Implications for H2 Production

Formate-dependent proton reduction to H₂ (HCOO⁻ + H₂O → HCO₃⁻ + H₂) has been reported for hyperthermophilic Thermococcus strains. In this study, a hyperthermophilic archaeon, Thermococcus onnurineus strain NA1, yielded H₂ accumulation to a partial pressure of 1 × 10⁵ to 7 × 10⁵ Pa until the values of Gibbs free energy change (ΔG) reached near thermodynamic equilibrium (−1 to −3 kJ mol⁻¹). The bioenergetic requirement for the metabolism to conserve energy was demonstrated by ΔG values as small as −5 kJ mol⁻¹, which are less than the biological minimum energy quantum, −20 kJ mol⁻¹, as calculated by Schink (B. Schink, Microbiol. Mol. Biol. Rev. 61:262-280, 1997). Considering formate as a possible H₂ storage material, the H₂ production potential of the strain was assessed. The volumetric H₂ production rate increased linearly with increasing cell density, leading to 2,820 mmol liter⁻¹ h⁻¹ at an optical density at 600 nm (OD₆₀₀) of 18.6, and resulted in the high specific H₂ production rates of 404 ± 6 mmol g⁻¹ h⁻¹. The H₂ productivity indicates the great potential of T. onnurineus strain NA1 for practical application in comparison with H₂-producing microbes. Our result demonstrates that T. onnurineus strain NA1 has a highly efficient metabolic system to thrive on formate in hydrothermal systems.     

Genetic and physical interaction of Ssp1 CaMKK and Rad24 14-3-3 during low pH and osmotic stress in fission yeast

The Ssp1 calmodulin kinase kinase (CaMKK) is necessary for stress-induced re-organization of the actin cytoskeleton and initiation of growth at the new cell end following division in Schizosaccharomyces pombe. In addition, it regulates AMP-activated kinase and functions in low glucose tolerance. ssp1⁻ cells undergo mitotic delay at elevated temperatures and G₂ arrest in the presence of additional stressors. Following hyperosmotic stress, Ssp1-GFP forms transient foci which accumulate at the cell membrane and form a band around the cell circumference, but not co-localizing with actin patches. Hyperosmolarity-induced localization to the cell membrane occurs concomitantly with a reduction of its interaction with the 14-3-3 protein Rad24, but not Rad25 which remains bound to Ssp1. The loss of rad24 in ssp1⁻ cells reduces the severity of hyperosmotic stress response and relieves mitotic delay. Conversely, overexpression of rad24 exacerbates stress response and concomitant cell elongation. rad24⁻ does not impair stress-induced localization of Ssp1 to the cell membrane, however this response is almost completely absent in cells overexpressing rad24.     

Expression of a novel gene, <Emphasis Type="Italic">gluP</Emphasis>, is essential for normal <Emphasis Type="Italic">Bacillus subtilis</Emphasis> cell division and contributes to glucose export

Background  

The Bacillus subtilis glucokinase operon was predicted to be comprised of the genes, yqgP (now named gluP), yqgQ, and glcK. We have previously established a role for glcK in glucose metabolism. In the absence of enzymes that phosphorylate glucose, such as GlcK and/or enzyme II^Glc, accumulated cytoplasmic glucose can be transported out of the cell. Genes within the glucokinase operon were not previously known to play a role in glucose transport. Here we describe the expression of gluP and its function in glucose transport.     

In glucose-limited continuous culture the minimum substrate concentration for growth,smin, is crucial in the competition between the enterobacterium Escherichia coli and Chelatobacter heintzii,an environmentally abundant bacterium

The competition for glucose between Escherichia coli ML30, a typical copiotrophic enterobacterium and Chelatobacter heintzii ATCC29600, an environmentally successful strain, was studied in a carbon-limited culture at low dilution rates. First, as a base for modelling, the kinetic parameters μ_max and K_s were determined for growth with glucose. For both strains, μ_max was determined in batch culture after different precultivation conditions. In the case of C. heintzii, μ_max was virtually independent of precultivation conditions. When inoculated into a glucose-excess batch culture medium from a glucose-limited chemostat run at a dilution rate of 0.075 h⁻¹ C. heintzii grew immediately with a μ_max of 0.17±0.03 h⁻¹. After five transfers in batch culture, μ_max had increased only slightly to 0.18±0.03 h⁻¹. A different pattern was observed in the case of E. coli. Inoculated from a glucose-limited chemostat at D=0.075 h⁻¹ into glucose-excess batch medium E. coli grew only after an acceleration phase of ∼3.5 h with a μ_max of 0.52 h⁻¹. After 120 generations and several transfers into fresh medium, μ_max had increased to 0.80±0.03 h⁻¹. For long-term adapted chemostat-cultivated cells, a K_s for glucose of 15 μg l⁻¹ for C. heintzii, and of 35 μg l⁻¹ for E. coli, respectively, was determined in ¹⁴C-labelled glucose uptake experiments. In competition experiments, the population dynamics of the mixed culture was determined using specific surface antibodies against C. heintzii and a specific 16S rRNA probe for E. coli. C. heintzii outcompeted E. coli in glucose-limited continuous culture at the low dilution rates of 0.05 and 0.075 h⁻¹. Using the determined pure culture parameter values for K_s and μ_max, it was only possible to simulate the population dynamics during competition with an extended form of the Monod model, which includes a finite substrate concentration at zero growth rate (s_min). The values estimated for s_min were dependent on growth rate; at D=0.05 h⁻¹, it was 12.6 and 0 μg l⁻¹ for E. coli and C. heintzii, respectively. To fit the data at D=0.075 h⁻¹, s_min for E. coli had to be raised to 34.9 μg l⁻¹ whereas s_min for C. heintzii remained zero. The results of the mathematical simulation suggest that it is not so much the higher K_s value, which is responsible for the unsuccessful competition of E. coli at low residual glucose concentration, but rather the existence of a significant s_min.     

Culturable Heavy Metal-Resistant and Plant Growth Promoting Bacteria in V-Ti Magnetite Mine Tailing Soil from Panzhihua,China

To provide a basis for using indigenous bacteria for bioremediation of heavy metal contaminated soil, the heavy metal resistance and plant growth-promoting activity of 136 isolates from V-Ti magnetite mine tailing soil were systematically analyzed. Among the 13 identified bacterial genera, the most abundant genus was Bacillus (79 isolates) out of which 32 represented B. subtilis and 14 B. pumilus, followed by Rhizobium sp. (29 isolates) and Ochrobactrum intermedium (13 isolates). Altogether 93 isolates tolerated the highest concentration (1000 mg kg⁻¹) of at least one of the six tested heavy metals. Five strains were tolerant against all the tested heavy metals, 71 strains tolerated 1,000 mg kg⁻¹ cadmium whereas only one strain tolerated 1,000 mg kg⁻¹ cobalt. Altogether 67% of the bacteria produced indoleacetic acid (IAA), a plant growth-promoting phytohormone. The concentration of IAA produced by 53 isolates was higher than 20 µg ml⁻¹. In total 21% of the bacteria produced siderophore (5.50–167.67 µg ml⁻¹) with two Bacillus sp. producing more than 100 µg ml⁻¹. Eighteen isolates produced both IAA and siderophore. The results suggested that the indigenous bacteria in the soil have beneficial characteristics for remediating the contaminated mine tailing soil.     

Effect of Light and NO(3) on Wheat Leaf Phosphoenolpyruvate Carboxylase Activity: Evidence for Covalent Modulation of the C(3) Enzyme

Phosphoenolpyruvate carboxylase (PEPcase) activity was studied in excised leaves of wheat (Triticum aestivum L.) in the dark and in the light, in presence of either N-free (low-NO₃⁻ leaves) or 40 millimolar KNO₃ (high-NO₃⁻ leaves) nutrient solutions. PEPcase activity increased to 2.7-fold higher than that measured in dark-adapted tissue (control) during the first 60 minutes and continued to increase more slowly to 3.8-fold that of the control. This level was reached after 200 minutes exposure of the leaves to light and high NO₃⁻. In contrast, the lower rate of increase recorded for low-NO₃⁻ leaves ceased after 60 minutes of exposure to light at 2.3-fold the control level. The short-term NO₃⁻ effect increased linearly with the level of NO₃⁻ uptake. In immunoprecipitation experiments, the antibody concentration for PEPcase precipitation increased with the protein extracts from the different treatments in the order: control, illuminated low-NO₃⁻ leaves, illuminated high-NO₃⁻ leaves. This order also applied with regard to a decreasing sensitivity to malate and an increasing stimulation by okadaic acid (an inhibitor of P-protein phosphatases). Following these studies, ³²P labeling experiments were carried out in vivo. These showed that the light-induced change in the properties of the PEPcase was due to an alteration in the phosphorylation state of the protein and that this effect was enhanced in high-NO₃⁻ conditions. Based on the responses of PEPcase and sucrose phosphate synthase in wheat leaves to light and NO₃⁻, an interpretation of the role of NO₃⁻ as either an inhibitor of P-protein phosphatase(s) or activator of protein kinase(s) is inferred. In the presence of NO₃⁻, the phosphorylation state of both PEPcase and sucrose phosphate synthase is increased. This causes activation of the former enzyme and inhibition of the latter. We suggest that NO₃⁻ modulates the relative protein kinase/protein phosphatase ratio to favor increased phosphorylation of both enzymes in order to redirect carbon flow away from sucrose synthesis and toward amino acid synthesis.     

Changes in phosphoinositide metabolism with days in culture affect signal transduction pathways in galdieria sulphuraria

The metabolism of phosphatidylinositol-4,5-bisphosphate (PIP₂) changed during the culture period of the thermoacidophilic red alga Galdieria sulphuraria. Seven days after inoculation, the amount of PIP₂ in the cells was 910 ± 100 pmol g⁻¹ fresh weight; by 12 d, PIP₂ levels increased to 1200 ± 150 pmol g⁻¹ fresh weight. In vitro assays indicated that phosphatidylinositol monophosphate (PIP) kinase specific activity increased from 75 to 230 pmol min⁻¹ mg⁻¹ protein between d 7 and 12. When G. sulphuraria cells were osmostimulated, transient increases of up to 4-fold could be observed in inositol-1,4,5-trisphosphate (IP₃) levels within 90 s, regardless of the age of the cells. In d-12 cells, the increase in IP₃ was preceded by a transient increase of up to 5-fold in specific PIP kinase activity, whereas no such increase was detected after osmostimulation of d-7 cells. The increase in PIP kinase activity before IP₃ signaling in d-12 cells indicates that there is an additional pathway for regulation of phosphoinositide metabolism after stimulation other than an initial activation of phospholipase C. Also, the rapid activation of PIP₂ biosynthesis in cells with already-high PIP₂ levels suggests that the PIP₂ present was not available for signal transduction. By comparing the response of the cells at d 7 and 12, we have identified two potentially distinct pools of PIP₂.     

Degradation of nanoRNA is performed by multiple redundant RNases in Bacillus subtilis

Escherichia coli possesses only one essential oligoribonuclease (Orn), an enzyme that can degrade oligoribonucleotides of five residues and shorter in length (nanoRNA). Firmicutes including Bacillus subtilis do not have an Orn homolog. We had previously identified YtqI (NrnA) as functional analog of Orn in B. subtilis. Screening a genomic library from B. subtilis for genes that can complement a conditional orn mutant, we identify here YngD (NrnB) as a second nanoRNase in B. subtilis. Like NrnA, NrnB is a member of the DHH/DHHA1 protein family of phosphoesterases. NrnB degrades nanoRNA 5-mers in vitro similarily to Orn. Low expression levels of NrnB are sufficient for orn complementation. YhaM, a known RNase present in B. subtilis, degrades nanoRNA efficiently in vitro but requires high levels of expression for only partial complementation of the orn^– strain. A triple mutant (nrnA^–, nrnB^–, yhaM^–) in B. subtilis is viable and shows almost no impairment in growth. Lastly, RNase J1 seems also to have some 5′-to-3′ exoribonuclease activity on nanoRNA and thus can potentially finish degradation of RNA. We conclude that, unlike in E. coli, degradation of nanoRNA is performed in a redundant fashion in B. subtilis.     

Molecular Characterization of a Glucokinase with Broad Hexose Specificity from Bacillus sphaericus Strain C3-41

Bacillus sphaericus cannot metabolize sugar since it lacks several of the enzymes necessary for glycolysis. Our results confirmed the presence of a glucokinase-encoding gene, glcK, and a phosphofructokinase-encoding gene, pfk, on the bacterial chromosome and expression of glucokinase during vegetative growth of B. sphaericus strains. However, no phosphoglucose isomerase gene (pgi) or phosphoglucose isomerase enzyme activity was detected in these strains. Furthermore, one glcK open reading frame was cloned from B. sphaericus strain C3-41 and then expressed in Escherichia coli. Biochemical analysis revealed that this gene encoded a protein with a molecular mass of 33 kDa and that the purified recombinant glucokinase had K_m values of 0.52 and 0.31 mM for ATP and glucose, respectively. It has been proved that this ATP-dependent glucokinase can also phosphorylate fructose and mannose, and sequence alignment of the glcK gene indicated that it belongs to the ROK protein family. It is postulated that the absence of the phosphoglucose isomerase-encoding gene pgi in B. sphaericus might be one of the reasons for the inability of this bacterium to metabolize carbohydrates. Our findings provide additional data that further elucidate the specific metabolic pathway and could be used for genetic improvement of B. sphaericus.     

The Tyrosine Kinase p56lck Mediates Activation of Swelling-induced Chloride Channels in Lymphocytes

Osmotic cell swelling activates Cl⁻ channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56^lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (I_Cl−swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of I_Cl−swell is defective in p56^lck-deficient cells. Retransfection of p56^lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of I_Cl−swell. Addition of purified p56^lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56^lck washed into the cytoplasm activates I_Cl−swell in native and p56^lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56^lck, slow single-channel gating events can be observed revealing a unitary conductance of 25–28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56^lck. We conclude that osmotic swelling activates I_Cl−swell in lymphocytes via the tyrosine kinase p56^lck.     

Photosynthetic carbon metabolism of a marine grass

The δ¹³C value of a tropical marine grass Thalassia testudinum is −9.04‰. This value is similar to the δ¹³C value of terrestrial tropical grasses. The δ¹³C values of the organic acid fraction, the amino acid fraction, the sugar fraction, malic acid, and glucose are: −11.2‰, −13.1‰, −10.1‰, −11.1‰, and −11.5‰, respectively. The δ¹³C values of malic acid and glucose of Thalassia are similar to the δ¹³C values of these intermediates in sorghum leaves and attest to the presence of the photosynthetic C₄-dicarboxylic acid pathway in this marine grass. The inorganic HCO₃⁻ for the growth of the grass fluctuates between −6.7 to −2.7‰ during the day. If CO₂ fixation in Thalassia is catalyzed by phosphoenolpyruvate carboxylase (which would result in a −3‰ fractionation between HCO₃⁻ and malic acid), the predicted δ¹³C value for Thalassia would be −9.7 to −5.7‰. This range is close to the observed range of −12.6 to −7.8‰ for Thalassia and agree with the operation of the C₄-dicarboxylic acid pathway in this plant. The early products of the fixation of HCO₃⁻ in the leaf sections are malic acid and aspartic acid which are similar to the early products of CO₂ fixation in C₄ terrestrial plants.     

Survival and differentiation defects contribute to neutropenia in glucose-6-phosphatase-β (G6PC3) deficiency in a model of mouse neutrophil granulocyte differentiation

Differentiation of neutrophil granulocytes (neutrophils) occurs through several steps in the bone marrow and requires a coordinate regulation of factors determining survival and lineage-specific development. A number of genes are known whose deficiency disrupts neutrophil generation in humans and in mice. One of the proteins encoded by these genes, glucose-6-phosphatase-β (G6PC3), is involved in glucose metabolism. G6PC3 deficiency causes neutropenia in humans and in mice, linked to enhanced apoptosis and ER stress. We used a model of conditional Hoxb8 expression to test molecular and functional differentiation as well as survival defects in neutrophils from G6PC3^−/− mice. Progenitor lines were established and differentiated into neutrophils when Hoxb8 was turned off. G6PC3^−/− progenitor cells underwent substantial apoptosis when differentiation was started. Transgenic expression of Bcl-X_L rescued survival; however, Bcl-X_L-protected differentiated cells showed reduced proliferation, immaturity and functional deficiency such as altered MAP kinase signaling and reduced cytokine secretion. Impaired glucose utilization was found and was associated with ER stress and apoptosis, associated with the upregulation of Bim and Bax; downregulation of Bim protected against apoptosis during differentiation. ER-stress further caused a profound loss of expression and secretion of the main neutrophil product neutrophil elastase during differentiation. Transplantation of wild-type Hoxb8-progenitor cells into irradiated mice allowed differentiation into neutrophils in the bone marrow in vivo. Transplantation of G6PC3^−/− cells yielded few mature neutrophils in bone marrow and peripheral blood. Transgenic Bcl-X_L permitted differentiation of G6PC3^−/− cells in vivo. However, functional deficiencies and differentiation abnormalities remained. Differentiation of macrophages from Hoxb8-dependent progenitors was only slightly disturbed. A combination of defects in differentiation and survival thus underlies neutropenia in G6PC3^−/− deficiency, both originating from a reduced ability to utilize glucose. Hoxb8-dependent cells are a model to study differentiation and survival of the neutrophil lineage.     

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca²⁺ entry (SOCE) with Orai1 as principal Ca²⁺ entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca²⁺ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1^−/− and Orai1^−/− platelets had greatly impaired glycoprotein (GP) VI-dependent Ca²⁺ signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2^−/− platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca²⁺ signals of Stim1^−/− and Orai1^−/− platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1^−/− and Orai1^−/− platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365, a blocker of (receptor-operated) Ca²⁺ entry, inhibited Ca²⁺ and procoagulant responses even in Stim1^−/− and Orai1^−/− platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca²⁺ entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca²⁺ entry and PS exposure, only one relying on STIM1-Orai1 interaction.     

Metabolic Engineering of Bacillus subtilis for Ethanol Production: Lactate Dehydrogenase Plays a Key Role in Fermentative Metabolism

Wild-type Bacillus subtilis ferments 20 g/liter glucose in 48 h, producing lactate and butanediol, but not ethanol or acetate. To construct an ethanologenic B. subtilis strain, homologous recombination was used to disrupt the native lactate dehydrogenase (LDH) gene (ldh) by chromosomal insertion of the Zymomonas mobilis pyruvate decarboxylase gene (pdc) and alcohol dehydrogenase II gene (adhB) under the control of the ldh native promoter. The values of the intracellular PDC and ADHII enzymatic activities of the engineered B. subtilis BS35 strain were similar to those found in an ethanologenic Escherichia coli strain. BS35 produced ethanol and butanediol; however, the cell growth and glucose consumption rates were reduced by 70 and 65%, respectively, in comparison to those in the progenitor strain. To eliminate butanediol production, the acetolactate synthase gene (alsS) was inactivated. In the BS36 strain (BS35 ΔalsS), ethanol production was enhanced, with a high yield (89% of the theoretical); however, the cell growth and glucose consumption rates remained low. Interestingly, kinetic characterization of LDH from B. subtilis showed that it is able to oxidize NADH and NADPH. The expression of the transhydrogenase encoded by udhA from E. coli allowed a partial recovery of the cell growth rate and an early onset of ethanol production. Beyond pyruvate-to-lactate conversion and NADH oxidation, an additional key physiological role of LDH for glucose consumption under fermentative conditions is suggested. Long-term cultivation showed that 8.9 g/liter of ethanol can be obtained using strain BS37 (BS35 ΔalsS udhA⁺). As far as we know, this is the highest ethanol titer and yield reported with a B. subtilis strain.