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Alcoholic Beverages : Wine, Vodka.Bakery & Confectionery Items : Chocolate, Cookies.Petroleum Products : Liquefied Natural Gas ( LNG ), Liquefied Petroleum Gas ( LPG ), Jet A1, Russian D2, Fuel Oil.Ppe, Electrical Transformer, Power Transformer, Buncker C, Cosmetic, Breakfast cereal, Snacks, Frozen Poultry, Urea 45 Fertilizer, Luxury Footwear, Etyl Alcohol, Ventilator, Thermometer, Sanitizer, Covid-19 Testing Kit,, Anticovid19film, Honey.
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Building Material : Cement Clinker.Garments : Used Shirts & Polo T-shirts.Bakery & Confectionery Items : Sweet Biscuits.Home Textiles & Furnishings : Cotton Bedsheets.Cosmetics : Perfumes, Disposable Face Mask.Minerals : Copper Ores, Beryl Ore, Raw Bauxite Ores.Dry Fruits : Cashew Nuts, Almond Nuts, Walnuts, Pistachio Nuts, Dried Apple Ring.Edible Oils : Refined Sunflower Oil, Corn Oil, Palm Oil, Soybean Oil, Peanut Oil.Food Products : Spaghetti, Spaghetti Pasta, Tomato Ketchup, Tomato Paste.Pulses & Lentils : Chickpeas, Red Split Lentils, Yellow Split Peas, Kidney Beans, Green Mung Beans, Chana Dal.Spices : Chili Pepper Seeds, Cumin Seeds, Black Pepper, White Pepper, Green Cardamom, Dried Clove, Red Chili Powder.Seafood : White Wild Shrimp, Black Tiger Shrimps, Dried Sea-cucumber, Ribbon Fish, Ocean Perch ( Red Fish), Mackerel, Crabs.Agricultural Commodities : Brown Parboiled Rice, Long Grain White Rice, Jasmine Rice, Basmati Rice, Brown Rice, Black Rice, Yellow Corn & Maize, White Maize, Red Millet.Meat & Poultry Products : Halal Frozen Boneless Buffalo Meat, Parrot Fertile Eggs, Halal Frozen Chicken, Feet, Whole Frozen Chicken Feet, Chicken Paws, Chicken Eggs.Fresh Fruits & Vegetables : Apples, Oranges, Grapes, Lemon, Avocado, Ginger, Garlic, Carrot, Onions, Bananas, Kiwi, Pomegranate, Tomatoes, Potatoes, Artichokes, Canned Lychee, Rambutan, Mandarin, Mangoes, Blueberries, Cabbages, Dragon Fruit, Green Beans.Alcoholic & Non-alcoholic Beverages : Coca Cola Soft Drinks, Monster Energy Drinks, Red Bull Energy Drink, Heineken Beer, German Beer & European Beer, Vodka, Fanta Soft Drinks, Fruit Juices, Coke Cans, Pepsi Range Cans, Red Bull Cans, Franziskaner Beer, Capri Sonne Juice, Nata De Coco Juice, Tsingtao Beer.Chemicals : Stearic Acid, Soda Ash, Caustic Soda, Potassium Chloride, Zinc Sulphate Monohydrate, Titanium Dioxide, Sodium Tripolyphosphate ( STPP ), Sodium Perborate Tetrahydrate, Sodium Percarbonate, Sodium Metasilicate, Citric Acid, Phosphoric Acid, Sodium Bicarbonate, Ascorbic Acid, Monoammonium Phosphate, Phosphoric Acid, Potassium Nitrate, Potassium Sulphate, Ammonium Molybdate, Ammonium Phosphate, Sorbic Acid, Sodium Hydrogen Sulphide, Sulphuric Acid, Ferrous Sulphate, Hydrated / Quick Lime, Methylene Chloride, Magnesium Sulphate, Acetic Acid, Sodium Ethyl Xanthate, Sodium Sulphate / Sulphide, Potassium Sorbate, Sodium Acid Pyrophosphate, Sodium Ascorbate, Sodium Benzoate, Sodium Cmc, Sodium Hexametaphosphate, Sodium Gluconate, Calcium Chloride, Sodium Hydroxide, Sulphonic Acid, Sodium Molybdate, Manganese Sulphate Monohydrate, Zinc Sulphate Heptahydrate.Iced Coffee Drink, Barbican Carbonated Drinks, Frozen Lingonberry, Canned Pineapple, Dried Papaya, Chia Seed Drink, Tea, Sweet Corn, Figs, Oat, Cotton Waste, Pecan nuts, Hazel Nuts, Dried Mango, Rapeseed Oil, Olive Oil, Canola Oil, Olive Oil, Hemp Oil, Coconuts, Hardwood Charcoal, Coconut Shell Charcoal, Coffee Beans, Wet Salted Donkey Hides & Skin, Sisal Fiber, Frozen Salmon Heads, Low Fat Yogurt, Palm Sugar, Whole Roasted Eggplant, Fridge Compressor Scrap, Aluminium Ingot, Aluminium Scrap, EPS Block, Stainless Steel, Saffron, Sesame Residue Powder, Hemp Seeds, Pumpkin Seeds, Sunflower Seeds, Safflower Seed, Hot Washed Pet Recycled Flakes, Waste Paper Scrap, Plastic Scrap, Sesame Seeds, Full Cream Milk Powder, Skimmed Milk Powder, Infant Baby Milk, Powder, Dried Garlic Flakes, Soy Sauce Powder, Raw Cotton, Guava Juice Concentrate, Banana Juice Concentrate, Pawpaw Juice Concentrate, Pineapple Juice Concentrate, Kiwi Fruit Juice Concentrate, Mango Juice Concentrate, Lemon Juice Concentrate, Lime Juice Concentrate, Grapefruit Juice Concentrate, Polymethyl Methacrylate, Liquid Silicone Rubber, Sodium Saccharin, Toilet Flush Valve, Fresh Button Mushroom, Yeast Extract Powder, Ginger Powder, Avocado Powder, Corn Flakes, Canned Foods, Macadamia Nuts, Pine Nut kernels, Mixed Nuts, Seasonings, Chia Seeds, Brazil Nuts, Cottonseed Hull Pellets, Frozen Yogurt Powder, Second Hands Big Bags, Raw Redskin Peanuts, Soy Milk Powder, Nutrilon Infant Milk Powder, Carrot Juice Concentrate, Popcorn Mushroom Kernels, Used Vegetable Cooking Oil, Soy Fatty Acid, Coconut Oil, Flour, Corn Gluten Meal, Iron Powder, Canned Soybeans Sprouts, Canned Corned Beef, Mushroom, Bamboo Shoots, Pecan Nut Kernels, Lotus Seeds, Evaporated Milk Powder, Sweetened Condensed Milk, Vanilla Beans, Whey Protein Concentrate, Popcorn Kernels, Semi-husked Mature, Sorghum Grain Meal, Chestnuts, Raw Cashew Nuts, Hazelnuts, Peanut butter, Fish Meal, Alfalfa Hay, Soybean Meal, Sunflower Seed Meal, Wood Pellet, Salted Peanuts, Canned Baked Beans, Sweet Potato Starch, Sugar Icumsa 45, Corn Starch, Butter Beans, Urea, Roasted Buckwheat, Meat Bone Meal, Fresh Bbq Charcoal, Alkalized Cocoa Powder, Wheat Grain, A4 Copy Paper, Native Tapioca Starch, Human Hair Extensions, Pure Natural Honey, Canned Fruit Cocktails ( Tropical), Confectionery, Rough Diamonds, Copper, Ferro Silicone Manganese, Animal Feeds, Cashew Nuts Shell Liquid, Clear Solvent Low Aromatic Industrial White Spirit, Cattle Fodder, Anhydrous Milk Fat, Green Millet ( Bajra ), Wheat Straw, Wheat-Bran, Beet Pulp, Electronics / Plasma Television, Blood Meal, Fish Meal, Grain, Hay, Milk Powder, Peanut Powder, Salt Lick, Seaweed Powder, Seedlings, Soybeans, Vegetable Powder, Worm, Corn Gluten Meal, Millet Meal, Rice-Bran, Wheat-Bran, Alfalfa Hay, Grain Hay, Mixed Hay, Timothy Hay, Cotton Seed Meal, Hemp seeds, Rapeseed meal, Tea Seed Meal, Earthworm, Mealworm, Silkworm, Refined Grease Oil, MC FeMn, Lithium Grease, Tinplate Beer Ice Bucket, Freeze Dried Fruit, Masafi Flavoured Water, Glucose Syrup & Corn Syrup, Tropik Splash Juice, Instant Tea Powder, Corn Flakes & Chocolate Coated cereals, Sliced Jalapenos, Milk Shakes, Sella, Hulled Buckwheat, Canned Sweet Corn, Canned Sweet Peas, Canned Cut, Hard Milling Wheat, Soya Meal & Corn Silage, Crushed Tomato, Skin Pomelo Grapefruit, Oat Flakes, Rice Flakes, Almond Flakes, Young Green Jackfruit, Frozen Grilled Zucchini & Pineapple, Dried Fig, Chocolate Drink Powdelinseed, White Bentonite Clay, Glycerin, Paraffin, Ostrich Chicks, Urea Phosphate, Dyes, Glycerine, Np9, Potassium Hydroxide Flakes, Sodium Cmc, Sles, Sodium Cmc, Propylene Glycol, Castor Oil First Pressing, Glycerol Mono Stearate 40/90, Guargum F.g, Maize Starch, Potato Starch, Coffee, Cocoa, Cocoa Powder, Mineral Oil Light / Heavy, Mono Propylene Glycol Usp, Potassium Sorbate Granular/powder, Xanthan Gum, Industrial Sea Salt. Acesulfame K, Acetic Acid Glacial, Aspartame, Calcium Chloride F.g, Citrus Pectin, Guargum F.g, Sodium Metabisulphite F.g, Sodium Saccharin, Sorbitol, Soya Lecithin F.g, Whey Powder, Xanthan Gum, Hydrogen Peroxide, Lithium Carbonate / Hydroxide, Aluminium Sulphate Granular, Melt Blown Fabric, Hydrobromic Acid, Ostrich Chicks and Eggs, Old Phone Scraps, Cow Gallstone, Ostrich Products, Chebe Powder, Catalytic Converter Scrap.
Seafood : Fish.Dairy Products : Milk.Agricultural Products : Rice.Spices : Pepper, Black Pepper.Livestock : Goats, Cows, Sheep Goats.Tobacco Products : Herbal Cigars & Cigarettes.Medical Items : Ir Non-contact Thermometers.Security & Surveillance Equipment : CCTV Camera.Confectionary Items : White Chocolate Almonds Bark.Dry Fruits : Cashew Nuts, Pistachio Nuts, Almond Nuts, Walnuts.Fresh Fruits & Vegetables : Blueberry, Cherries, Apples, Bananas, Water Melon, Beans.Meat & Poultry Products : Pork, Meat, Chicken, Chicken Flaws, Chicken Wings, Chicken Feets, Beaf, Eggs & Eggs Products.Alcoholic Beverages, Baby Food, Baked Goods, Canned Food, Coffee, Confectionery, Dairy, Drinking Water, Food Ingredients, Fruit Products, Grain Products, Honey Products, Instant Food, Seafood, Seasonings & Condiments, Snack, Drinks, Locks & Keys, Personal Protective Equipment, Control Systems & Products, Alarm, Fire Fighting Supplies, Locksmith Supplies, Security & Protection Products, Police & Military Supplies, Roadway Safety, Defense Supplies, Water Safety Products, Bullet Proof Vest, Alcohol Tester, Fire Alarm, Coconut, Consumer Electronic, Computer Hardware & Software, Home Audio, Video & Accessories Mobile Phone &, A4 Copy Papers, Alcoholic & Non-alcoholic Beverages, Farm Products, Processed Food, Processed Food, Cashews, Animal Products, Fish Products, Coconut Shell Products, Olive Oil, Car Tyres / Tires, Motor Parts, Passenger Cars, Fresh Flowers, Coal, Fertilizers And Pesticides, cereal, Animal Fodders, Mushroom And Truffle, Dry Fruits, Seedlings, Tea And Coffee, Spices, Face Mask, Gloves, Sanitizers, Surgical Mask, Pharmaceutical Products, Used Cars, Protective Masks, Eye Mask, Protective Cap, Disinfectant, Hand Sanitizer, Medical Alcohol, Eye Protection, Disposable Latex Gloves, Protective Suit, Human Body Thermal Cameras, Face Masks, Used Tyres / Tires, Seafood, Brazilian Nuts, Pine Nuts, Peanuts, Groundnuts, Pumpkins Spice, Californias Figs, Baobab Pomegranate Bites, Green Tea, Ingots, Scraps, Coffee, Computer Scraps, Plastic Scraps, Paper Scraps, A Copy Paper, Copy Paper, Igg/igm Rapid Test Kit, Gloves, Iron Scraps, New Tyres / Tires, Flakes, Cattle, Coconuts, Goats, Sheep Cattle, Used Tires, 250-200 Hp 4.2l V Max Sho Outboard Motors, Brand New / Used 2018 Yamaha 150hp Outboards Motors, Palnd And Animal Oil, Oil, Engine Oil, Scraps, Agricultural Waste, Animal Products, Cocoa Beans, Coffee Beans, Farm Machinery & Equipment, Feed, Grain, Nuts & Kernels, Ornamental Plants, Plant & Animal Oil, Plant Seeds, Bulbs, Timber, Raw Materials, Vanilla Beans.
Fresh Vegetables : Beans.Agricultural Products : Rice.Poultry Products : Pork, Beef, Meat.Disposable Face Masks 3ply, Ffp2 / Kn95 Face Masks, Covid-19 Test Kits, Air Sanitizer, Waste Management, End Of Life Tires System, cereals, Seedlings.
Petroleum Products : Crude Oil, Bonny Light Crude Oil ( Blco ), Russian Light Crude, Diesel / D2, Diesel / D6, Diesel Ultra Low Sulphur Diesel (ULSD ), Diesel En590, Jp54 Jet A191/91 (aviation Kerosene Colonial Grade 54), Russian Hsd2 50ppm, Russian Export Blend Crude ( REBCO ) Gost 51 858-2002/9965-76, Hsd2 Gas Oil, Liquefied Petroleum Gas ( LPG ), Liquefied Natural Gas ( LNG ), Bitumen, Russian Mazut M100, Gost 10585-75/10585-99, Premium Motor Spirit (PMS), Russian Cst - 180/380 Fuel Oil, Gasoline 91 Ron.Seafood : Natural Fillets, Cut Portions, Whole Round Fish.Agricultural Products : Sella / Parboiled And Polished & Golden Sella Rice, Corn. Pulses & Lentils : Speckled Green, Black, Red, Yellow & Green Beans, Chickpeas.Prefabricated Low Energy Structures, Industrial Equipment & Turnkey Industrial Projects, Biodiesel Plants, Ethanol Plants, Uco Refineries, Dap Di - Ammonium.phosphate, Diammonium Phosphate, Russian Base Pilot, Pet Coke, Sulphur Granular, Compressed Natural Gas (cns), Grain, Gmo & Non Gmo Soybeans, Soybean Meal, Soy Hulls, Wheat Corn ( For Human Consumption, Animal Feed, Ethanol Production, High Fructose Corn Syrup Production, Bio Based Plastic Production), Brown, Raw, Steam Rice, Pulse & cereals, Raw & Refined Sugar (white & Brown), Meat & Poultry, Lecithin, Rapeseedmeal, Soybean Meal, Feed Barley, Feed Grains, Crude Vegetable Oil (sunflower, Rapeseed, Flaxseed, Camellia, Safflower, Soybean, Fat Acid Oils), Fresh & Frozen Vegetables & Fruits.
Poultry Products : Eggs.Religious Items : Agarbatti.Pulses & Lentils : Chickpeas.Fresh Vegetables : Beans.Dry Fruits : Almonds.Alcoholic Beverages : Beer.Poultry Products : Beef.Spices : Black Pepper, Cinnamon,Cardamom, Petroleum Products : Bitumen.Home Furnishings : Blankets, Carpets.Stationary Items : Books.Sports Goods : Baseballs.Bakery Items : Biscuits.Petroleum Products : Bonny Light Crude Oil ( Blco ).Electrical / Home Appliances : Air Conditioning.Security & Surveillance Equipment : Cctv Camera.Abalone, Abs, Acetic Acid, Acp, Adapters, Adsl, Agar, Agricultrual Machinery, Agricultural Commodities, Agricultural Machines, Agro-chemicals, Air Freight, Aircrafts, Alcoholic Beverages, Alexandrite, Alfalfa, Alluminium, Aluminium Ores, Alluvial Dust, Aluminum, Aluminum Composite Panels, Aluminum Ores, Aluminum Panel, Amethyst, Anchovy, Ani Seeds, Animal Feed, Aniseeds, Anklets, Antibiotics, Antique, Apparels / Garments, Appliances, Apricots, Aquaculture, Auto Part, Auto Rickshaw, Baskets, Bathrobes, Bathtubs, Battery, Bauxite, Beautycare, Beewax, Bentonite, Betel Nuts, Bgs, Bicycles, Billets, Bioriented Polypropylene, Bird Houses, Bird Nest, Block Board, Blood Meal, Bodycare, Bolts, Bone Meal, Bopp, Briefcases, Building Materials, Bulbs, Bulldozers, Burners, Butter, Cages, Calculators, Camera, Candle Holder, Candy, Canvas, Car Parts, Carbon Steel, Card Readers,Cartons, Cartridges, Cashews, Cattles, Caustic Soda, Cbs, Cdma, Ceiling, Cell Phones, Cellulose, Cement, cereal, Chairs, Champaign, Charcoal, Chemical Raw Materials, Chewing Gum, Cigarettes, Cigars, Circuit Boards, Citric Acid, Clamps, Coconut, Compactors, Condensers, Cookers, Copier Paper, Corn, Dairy, Dispensers, Dragon Fruits, Drugs, Dry Fruits, Dvd, Earrings, Edible Oil, Electrical, Engine Parts, Escalators, Ethernet, Excavators, Eyewear, F12, F22, Fabrics, Fertiliser, Fish, Flower Pots, Footwear, Furniture, Gifts, Grains, Guava, Hacksaw, Hair Accessories, Hair Products, Hairpins, Hammers, Harrows, Harvester, Home Appliances.
Food Products : Snacks.Building Material : Cement.Minerals : Magnetite Iron Ores.Confectionery Items : Chocolates.Dairy Products : Milk & Milk Products.Non-alcoholic Beverages : Energy Drinks.Pulses & Lentils : White Kidney Beans, Black Beans.Dry Fruits : Cashew Nuts W320, Walnuts, Pistachios.Chemicals : Chloride Potassium, Monocalcium Phosphate.Spices : Green Cardamom, Penja White Pepper, Dry Ginger.Poultry Products : Chicken & Chicken Paw, Sheep/Goat Meat, Eggs.Hygiene Products : Diapers, Dettol Hand Sanitizer, Facial Wet Wipes.Agricultural Products : Corn, Barley, Basmati & Non-basmati Rice, Wheat.Plastic Raw Material : High-density Polyethylene ( HDPE ), Low-density Polyethylene ( LDPE ).Poultry Products : Egg & Egg Products, Meat, Chicken Eggs, Ostrich Chicks, Ostrich Eggs.Home Textile & Furnishing : Mattresses, Quilts, Towels, Flooring, Cushions & Pillows, Bedding Set, Towel, Chair Cover.Electrical / Home Appliances : Air Conditioning, Refrigerators, Washing Machines, Vacuum Cleaners.Pulses & Lentils : Light Speckled Kidney Beans, Lima Beans, Green Peas, Dried Red Kidney Bean, White Kidney Beans, Sugar Beans, Chickpeas, Green Mung Beans.Seafood : Frozen Octopus & Squid Tube, Block Frozen Skipjack Tuna Heads, Lacha Fish, Frozen Raw Or Cooked Crayfish, Fish & Prawns. Fresh Fruits & Vegetables : Beans, Garlic, Onion, Cavendish Banana, Red Dragon Fruit, Potatoes, Sweet Potato, Mangoes, Mangosteens, Guava, Cucumbers, Oranges, Watermelons, Carrots, Turnips, Lemons, Ginger, Green Grapes, Passion Fruit, Jackfruit, Mangosteen, Mushroom, Pomegranate.Non-agricultural Products, Seedling, Nuts, Agro-chemicals, Fertilizers, Pesticides, Minerals, Metal & Alloys, Sandstones & Sand, Clear Float Glass, Calcinated Bauxite, Soybean Meal, Raw Cashew Nuts, A4 Copy Paper 70 Gsm, Napkin Roll, White Petroleum Jelly, Mopcarded Ring Yarn, Steel Barrels, Stainless Steel, Blanched Peanuts, Yellow Rapeseed, Crystal And Green Bottle Flakes, Pringles, Soda Ash Dense, Aluminum Ingots, Alfalfa Hay, Coffee, Disposable Medical Mask, Softwood Pulp, Hand Bags / Sacks, Sugar, Plastic Raw Material, Sugarcane Syrup, Medical Equipment, Ginseng Liquid Extract Oil, Methanol Industrial Grade, Bauxite, Cotton Jersey Fabric, Coffee Cups, Popcorn Kernels, Plastic Bottles, Timber / Wood, Liquid Helium, Iqf Pineapples Diced, Crystal Salt Non Iodized, Quartz Powder, Bio Fuel, Cotton Yarn, Cooking Oil, Sugarcane Products, Inconel 600 Seamless Pipes & Fittings, Palm Kernel, Limestone, Vegetable Fatty Acid Distillate ( Fad ), A4 Copier Paper, Toilet Papers & Rolls, Anthracite Coal, Cooking Coal, Coconut Copra, Coconut Shell Charcoal, Canola Seeds, Soybeans Animal Feed, Dry Dog Food, Semolina Flour, Flour Bonkalit, Meat Products, Disposable Medical Mask, Apron, Fertilizers, Shredded Tyres, Processed Meat, Animal Casings, Fruits / Vegetable-seeds, Groundnut, Processed Fruits & Juices, cereal Preparations, Guargum, Misc Processed Items, Alcoholic Beverages, Processed Vegetables, cereals, Pulses, Cocoa Products, Milled Products, Floriculture, Cattle, Raw Cotton, Sugar, Cassava, Coffee, Rubber, Tea, Lime Salt, Starches, Insulin, Wheat Gluten, Soda Ash Dense, Garnet Abrasive Grit, Dried Hibiscus Flower, Biodegradable Bags, Vanilla Beans, Penicillin, Cephalosporin, Thienamycin, Erythromycin, Vancomycin, Streptomycin, Centamycin, Mupirocin, Daptomycin, Amphotericin B, Pneumocandin, Avermectin, Cyclosporin A, Tacrolimus, Rapamycin, Mycophendic, Streptococcus Mutans, Colon Cancer Antigen, Herpes Simplex Virus (hsv-2), Cattle, Corn, Raw Cotton, Dairy Products, Poultry Products, Soybeans, Sugar, Carcinogenic Antigen (cea), Non-hodgkins (nhl) Lymphoma, Carcinoembryonic Antigen (cea), Neofuragina, Neil Med Sinus Pionse, Keladerm, Alantan, Nesojie Spray, Vagisan, Vanicream, Uridrine 300mg, Metafen, Stimerex-es, Omega-3 Gold, Niverosin, Sovereign Silver, Clinipharma, Evaporating Dish, Dropper, Pipette, Bunsen Burner, Filter Paper, Burette, Stopper, Rubber Tubing, Adson Dressing Forceps Teeth, Adson Dressing Forceps Serrated, Artery Forceps Extra Large, Ayer Chalazion Forceps, Balfour Retractor Standard Pattern Lateral, Blades, Ballenger Swivel Knife, Biopsy Forceps Chevalier Jack Son, Biopsy (uterine Van-doren Forceps), Biopsy (uterine Schubert Forceps), Blake Uterine Curettes Double Ended, Bone Chisels, Bone Chisles Stile, Bone Cutting Forceps Liston, Bone Guages, Bone Saws Satterlee, Bowman Dilators Set, Bozeman Uterine Dressing Forceps, Bryant�s Dressing Forceps, Oatmeal Oats, Quinoa, Organic Pumpkin Seeds, Nuts, Pecan Nuts, Macadamia Nuts, Betel Nut, Dried Whole Betel Nuts, Bitter Kola, Garcinia Kola-bitter Kola, Kola Nut, Sunflower Seeds, Brazil Nuts, Raw Hazelnut Roasted, Vietnam Macadamia Nuts, Wild Pine Nuts, Hazelnut, Alfalfa Seeds, Guarana Seed, Tomato Seeds, Paulownia Shantong, Sesame, Mombasa Grass Seeds, Hemp Seeds, Cucumber Seeds, Saffron Seed, Strawberry Seeds, Dried Lemon Seeds, Pao Tong Hot, Hemp Cbd Seeds, Mp171 Xuanhong High Yield Hot Red Chilli Pepper, Carrots, Dried Lotus Seed, Onion Seeds, Moringa Seeds, Vegetable Seeds, Fresh Natural Broccoli Seeds, 100pcs Dwarf Banana Tree Seeds, Papaya Seeds, F1 Green Cucumber Seed, Gumbo, Castor Oil Seeds, Fresh Soursoap,Hemp Extract Trichomes Pollen, Kief Flower Powder, CBD Crystalline Isolate, Ox Gallstone,Egg Powder, Soap Noddles.
Furniture : Chairs.Footwear : Shoes.Cosmetics : Face Mask.Fasteners : Nuts & Bolts.Edible Oils : Sunflower Oil.Agricultural Products : Rice.Tobacco Products : Cigarette.Seafood : Fish & Fish Products.Non-alcoholic Beverages : Soft Drink.Stationary Items : Books & Magazines.Home Textile & Furnishings : Towels, Carpets, Flooring, Cushions & Pillows, Mattresses, Quilts.Agricultural Products, Food Products, Alcoholic & Non-alcoholic Beverages, Processed Food, Farm Products, Grains, Fresh Fruits, Poultry & Other Animal Products, Coconut Shell Products, Cashews, Fertilizers & Pesticides, Seedlings, Tea & Coffee, cereals, Fresh Flowers, Vegetables, Pulses, Animal Fodders, Bamboo & Rattan Products, Dry Fruits, Mushroom & Truffle, Spices, Personal & Entertainment Products, Hand Bags & Suitcases, Music, Cosmetics, Toiletries & Hygiene Products, Handicrafts, Gifts & Decoratives, Gems & Jewellery, Home Textiles, Garments, Fashion Accessories, Toys, Games, Batteries, Beauty Care, Fabrics, Health & Baby Care, Computers & Electronics, Computer Software & Hardware, Electronics & Communications, Network Engineering, Notebooks & Laptops, Keyboards, Modems, Monitors & Displays, Printers & Scanners, Software, Amplifiers, Cd Players, Dvd Players, Mp3 Players, Radio & Television, Refrigerators, Washing Machines, Vacuum Cleaners, Bulbs & Tubes, Circuit Boards, Insulation Household & Furnishing, Heating & Air Conditioning, Furniture & Interior Decoration, Glass & Ceramics, Handkerchiefs, Clocks & Watches, Household Utensils, Kitchenware, Carvings, Briefcases & Suitcases, Auto / Automotive, Industrial Machinery & Plants, Marine Equipment, Metal & Metal Products, Motor Parts, Brakes, Chemicals, Packaging, Metal & Metal Products, Dyes, Varnish, Engine Parts, Tires / Tyres, Motorcycles & Parts, Minerals & Ores, Copy Paper & Paper Products, Petroleum & Petroleum Products, Plastic Products, Refrigeration & Storage, Rubber & Rubber Products, Auto Parts, Motor Parts, Used Rails, Bullion Gold, Diamond, Copper Cathode, Heineken, A4 Copy Paper, Sesame, Seedlings, Chia Seeds, Heineken, Used Rails Scrap, N95 Facemask, Cosmetics, 3ply Face Mask, Surgical Mask.
Seafood : Fish.Audio Products : Amplifiers.Tobacco Products : Cigarettes.Computer Peripherals : Notebooks & Laptops, Keyboards.Electrical / Home Appliances : Refrigerators, Washing Machines, Vacuum Cleaners.Agricultural Products, Food Products, Alcoholic & Non-alcoholic Beverages, Fish Products, Processed Food, Farm Products, Grains, Fresh Fruits, Poultry Products, Animal Products, Dogs, Cats, Horses, Parrots, Coconut Shell Products, Cashews, Fertilizers & Pesticides, Seedlings, Tea & Coffee, cereals, Fresh Flowers, Fresh Vegetables, Pulses, Animal Fodders, Bamboo & Rattan Products, Dry Fruits, Mushroom & Truffle, Spices, Personal & Entertainment Products, Hand Bags & Suitcases, Music, Cosmetics, Toiletries & Hygiene Products, Handicrafts, Gifts & Decorative, Gems & Jewellery, Home Textiles, Garments, Fashion Accessories, Toys, Games, Batteries, Beauty Care, Fabrics, Health & Baby Care, Computers & Electronics, Computer Software & Hardware, Electronics & Communications, Network Engineering, Modems, Monitors & Displays, Printers & Scanners, Software, Cd Players, Dvd Players, Mp3 Players, Radio & Television, Bulbs & Tubes, Circuit Boards, Insulation Household & Furnishing, Heating & Air Conditioning, Furniture & Interior Decoration, Glass & Ceramics, Handkerchiefs, Clocks & Watches, Household Utensils, Kitchenware, Carvings, Briefcases & Suitcases, Auto / Automotive, Industrial Machinery & Plants, Marine Equipment, Metal & Metal Products, Motor Parts, Brakes, Chemicals, Packaging, Metal & Metal Products, Dyes, Varnish, Engine Parts, Tires / Tyres, Motorcycles & Parts, Minerals & Ores, Copy Paper & Paper Products, Petroleum & Petroleum Products, Plastic Products, Refrigeration & Storage, Rubber & Rubber Products, Auto Parts, Motor Parts, Used Rails, Bullion Gold, Diamond, Copper Cathode, Heineken, A4 Copy Paper, Sesame, Seeds, Chia Seeds, Heineken, Used Rails, Surgical Mask, Ear lop Masks, N95, Betel Nuts.
Footwear : Shoes.Minerals : Iron Ore.Edible Oils : Sunflower Oil.Agricultural Products : Rice.Livestock : Saanen Goats, Cows, Buffalos.Dry Fruits : Cashew Nuts, Cashew Kennels.Fresh Vegetables : Onions, Beans, Mushroom.Alcoholic & Non-alcoholic Beverages : Red-bull, Energy Drinks, Heineken Beer, Coca Cola.Tea, Gallstone, Old Corrugated Card Boards (occ), Waste Paper Scrap, Paraffin Wax, Rubber, Paper Products, Fertilizers & Pesticides, Animal Products, cereals, House Hold Utensils, Beech, Birch, Ash, Maple, White Oak, Red Oak, Poplar, Pine, Spruce, Fir, Timber / Wood, Soybeans, Coco, Coffee, Seafood, Sugar, Copper Scrap, Copper Wire, Copper Cathode, Aluminum, Zinc, Used Rails, Hms 1&2 Scrap, Bullion Gold, Diamond, A4 Copy Paper, Copy White Paper, Used Cloths, Sport Supplements. Face Mask, 3m 1860, V Gloves, Nitrile Gloves, 3 Ply Face Mask, Pharmaceutical, Drugs, Pills, Bullion Gold, Gold Bars.
Agricultural Products : Millets.Pulses, cereals, Organic Dry Fruits, Brown Sugar, Blended Spices, Organic Salt, Jaggery.
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text: Photography is my main means for pursuing creative, artistic urges; for capturing and documenting live events; and for bringing joy to others through images of loved ones. I have photographed weddings, engagements, and other events. The people, the clothes, the ceremonies and celebrations, the flowers, the food, the beautiful settings...all come together to present a once in a lifetime event that most want to record in order to peruse and enjoy again and again throughout their lives. My goal in covering a wedding is to make photos that bring it all back to you...and to help you see parts you may have missed in the "whirlwind" of a busy and exciting day! My style is primarily "photojournalistic"; I'm trying to capture authentic expressions, activities, and emotions so you get the true flavor of the day's events. At the same time, I'm looking for artistic, symbolic imagery that gives your wedding a transcendent quality you can reflect on whenever you think of your partner and the sublime commitment you made to each other. My job is to make your wedding--through eloquent, expressive, and revealing imagery--last more than a day, but instead, a lifetime.

subset: commoncrawl


text:
3 Minute Kettlebell Sequence with 24KG
ContraGym Coach Bobby Thatcher doing a 3 Minute Kettlebell Sequence.Read more
ContraGym Coach Bobby Thatcher doing a 3 Minute Kettlebell Sequence.Read more
Tabata Workout – Kettlebell Tabata – Tabata Fitness Get the "Fat Decimator System" – http://cd4617p3lccxqt85o5naogznc7.hop.clickbank.net/ Get "Flat Belly Fix" – http://34508-14dcl3iv3bo4ondr4m3z.hop.clickbank.net/ Get "The 2 Week Diet" – http://bf39cvy2nbj6wofr90shq4jp66.hop.clickbank.net/ 20 sec Kettlebell Swing (Russian) 10 sec Rest 20 sec Kettlebell Side to Side 10 sec Rest 20 sec Kettlebell Snatch 10 sec Rest 20 sec Kettlebell…Read more
Kettlebell Training: Interval workout for fat loss & strength Jason Yun performs a quadset of :30 each for 4 rounds. This was #3 of 5. The kettlebell is a competition bell from Perform better and is 28 kg. To order a kettlebell or medicine ball from perform better visit this website: http://www.anrdoezrs.net/gq121cy63y5LTURUSSSLNOOMMUUULNQPMVPSOQSMMM The workout is…Read more
Finish 3 rounds of the given exercises in a circuit manner. Rest 120sec between rounds. Please note that this is an extreme level workout suitable only for people in superb shape! Get Your Interval Timer here; http://www.gymboss.com?src=IAP How to get in SHAPE? The right way! Let me help You get in the best shape of…Read more
Day one of our fat loss with Kettlebell Sport work. This is a great way to decrease fat while keeping muscle and using kettlebell sport to increase your endurance as well! -~-~~-~~~-~~-~- Please watch: "50 rep set of Double 106lb Suitcase Style Kettlebell Swings" https://www.youtube.com/watch?v=k85w51C16T0 -~-~~-~~~-~~-~-Read more


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Minot AFB gearing up for major construction season
MINOT AIR FORCE BASE – Assuming funding holds, it's going to be a huge year for larger facilities sustainment, restoration, modernization and investment to Minot Air Force Base infrastructure, says the commander of the base's 5th Civil Engineer Squadron.
"If we're able to execute and if funding holds, this could be the largest investment infrastructure we've had in the history of this base – close to $100 million in facilities sustainment, restoration and modernization (FSRM) funding," Col. Matthew Altman told members of the Minot Area Chamber of Commerce's Military Affairs Committee at its March meeting at Minot AFB.
In addition to a military construction project awarded last year and the Helicopter Operations and Tactical Response Force facility that will be awarded in a short time (estimated award is June), Altman said, "All total, 170-some million dollars potential pumped into the local economy this year alone – it's huge and we're really excited about it."
Looking back at projects completed in 2019, Altman listed those including:
® Medical Clinic, $38 million. Complete renovation. Completed in December 2019.
® Bomber Boulevard, $6.1 million. Full depth repair of majority of primary industrial thoroughfare. Completed October 2019.
"In addition to Bomber Boulevard, we've been painting all over this base to improve our infrastructure," Altman said. He said more painting will be done this year.
® Security Forces Training Center, $500,000. Renovation of classroom facility with funding from the 219th Security Forces Squadron of the North Dakota National Guard. Completed August 2019.
Paving projects completed from July 2018-November 2019 total more than $2.7 million.
Projects mainly completed but with some work remaining include:
® Aircraft parts building, $249,000. Renovation. Estimated complete date was February.
® Indoor activities/athletic center, $284,000. The year-round fitness and recreation activities facility, "The Turf," was completed and opened in February 2019.
"We renovated a warehouse with FY18 (fiscal year 2018) money for an indoor play facility," Altman said. "What we're excited about now is the future.
He said plans are to install an inflatable dome structure that would house a 300m track with field. Artificial turf would replace the grass field.
"We're going to copy what you have downtown and put up an air-inflated structure on a newly configured track with field surface and cover it with a dome," he said. "We're pretty confident this is going to execute. It is hoped the project can be awarded the first part of next year," Altman said.
He said such a facility will be a tremendous capability for the community – all the airmen and their families. He said the dome would be up about eight months of the year.
The inflatable dome structure is a fiscal year 2019-funded design and a fiscal year 2021 FSRM project for field and dome support.
® Mass parking apron. Third phase of the 60-year-old original concrete ramp, a seven-phase total $100 million project. The third phase has a $7.9 million estimated cost. The project is to be awarded this spring.
Editor's Note: The Department of the Army awarded Strata Corp., Grand Forks, a $9.5 million contract for the removal and replacement of about 23 square yards of concrete on the mass parking apron Section III, Senators Kevin Cramer and John Hoeven announced on Thursday and Friday. The estimated contract completion date is Oct. 29.
Current construction projects include:
® Welcome Center, $389,000. Former transient living facility being renovated for a Welcome Center – a one-stop shop for new airmen. Estimated completion date is April.
® B-52 maintenance dock renovation – Dock 6. $7.5 million. Replacement of infrastructure systems, building insulation and roof replacement. Estimated completion date is May.
® Indoor firing range construction, $23.4 million. Awarded in 2019 and ground breaking in August. Estimated completion date June 2021.
® Fitness Center upgrade, $1-5 million. Heating, ventilation, air conditioning and lighting. HVAC fiscal year 2020 completion; lighting out for bid, fiscal year 2021.
"This is the year of opportunity in my mind," Altman said of future construction.
Future construction include:
® B-52 maintenance dock renovation – Dock 3, $15-20 million, and Dock 5, $5-10 million. Estimated award is July.
® 91st Missile Wing Operations/Maintenance facility renovation, primarily maintenance side, $20-25 million. Two-phase project. $20-25 million. Phase 1 to upgrade existing industrial areas. Estimated award in 2020. Phase 2 includes renovation of 91st Missile Wing headquarters. Fiscal year 2022 design and fiscal year 2023 execution.
® Dakota Inn dining facility roofing replacement project, $1-3 million. Fiscal year 2020 funded; begin construction 2020.
® Paving projects. Total $1.5-2.5 million.
® Helicopter Operations and Tactical Response Force Facility, $60-70 million. Construct nine-bay facility to house new MH-139A Grey Wolf helicopters and tactical response force. Estimated award is June.
® Special Vehicle MX facility, $1-5 million. Construct pull-through vehicle maintenance facility to service Joint Land Tactical Vehicle, a vehicle expected to arrive in the next years. Fiscal year 2020 UMMC (unspecified minor military construction) project. 35% design complete.
Altman said currently, none of the facilities have doors large enough to drive in that massive vehicle.
® Renovate Child Development Center. $2-6 million. Convert old housing office for childcare space. Awaiting funding approval but expected to be funded in the fiscal year 2021 program.
Altman said the Child Development Center project will give four new classes for child development spaces.
5TH CE Squadron
The 5th Civil Engineer Squadron is comprised of 471 members – 329 military members and 142 civilians. Fire Emergency Services, Readiness and Emergency Management, and Explosive Ordnance Disposal are also part of the squadron. As of early March, 70 engineers from the squadron were on deployments in various areas in the world.


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Bring casual luxury to your table with this stunning Goa cutlery range from Cutipol. Crafted from high quality 18/10 stainless steel, this flatware range is dishwasher safe and comes individually wrapped in Cutipol tissue paper. Bringing contemporary style to any dining table, each item features a sleek, modern design with a black resin handle. The Goa flatware range is a fabulous gift idea for any occasion and looks beautiful paired with the wide range of Cutipol cutlery to create your own unique table setting.
Highest stainless steel with brushed finish sleek resin handle. The item "Cutipol Goa Cutlery Set -Black/Silver 24 Piece + Six Sets Chopsticks STUNNING" is in sale since Monday, January 22, 2018. This item is in the category "Home, Furniture & DIY\Cookware, Dining & Bar\Cutlery\Cutlery Sets & Canteens".
The seller is "tkawa01" and is located in London, London. This item can be shipped to all countries in Europe, United States, Japan, Canada, Australia.


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Ferdinand Gottschalk was born on February 28, 1858 in London. He was an English Actor, known for Grand Hotel (1932), The witching hour (1934), The Sign of the Cross (1932), Ferdinand Gottschalk's first movie on record is from 1917. Ferdinand Gottschalk died on November 10, 1944 in London. His last motion picture on file dates from 1937.
Ferdinand Gottschalk Filmography [Auszug]
1937: [] Carnival in Paris (Cast), Directed by Wilhelm Thiele, with Henry Brandon, Ann Rutherford, Harry Burns,
1935: [] Les Misérables (Cast), Directed by Richard Boleslawski, with Fredric March, Charles Laughton, Cedric Hardwicke,
1934: [11.14] The Man Who Broke the Bank at Monte Carlo (Cast: Office Man ), Directed by Stephen Roberts, with Ronald Colman, Joan Bennett, Colin Clive,
1934: [12.03] The Secret of the Chateau (Cast: Chief Inspector Marotte ), Directed by Richard Thorpe, with Claire Dodd, Alice White, Osgood Perkins,
1934: [10.05] Cleopatra (Cast: Glabrio), Directed by Cecil B. DeMille, with Claudette Colbert, , Henry Wilcoxon,
1934: [/ /1] The witching hour (Cast), Directed by Henry Hathaway, with George Webb, Selmer Jackson, Ernest Hilliard,
1934: [] I Sell Anything (Cast), Directed by Robert Florey, with Pat O'Brien, Ann Dvorak, Claire Dodd,
1934: [] King Kelly of the U.S.A. (Cast: King Maxmilian of Belgardia), Directed by Leonard Fields, with , Edgar Kennedy, Irene Ware,
1934: [03.31] Gambling Lady (Cast: Cornelius), Directed by Archie Mayo, with Barbara Stanwyck, Joel McCrea, Pat O'Brien,
1934: [01.19] Long Lost Father (Cast: The Lawyer), Directed by Ernest B. Schoedsack, with John Barrymore, Helen Chandler, Donald Cook,
1933: [07.29] Midnight Club (Cast: George Rubens), Directed by Alexander Hall, George Somnes, with Clive Brook, George Raft, ,
1933: [/ /1] Female (Cast: Pettegrew), Directed by Michael Curtiz, Wilhelm Dieterle, with Ruth Chatterton, George Brent, Johnny Mack Brown,
1933: [04.28] The Warrior's Husband (Cast: Sapiens Sr. ), Directed by Walter Lang, with Elissa Landi, David Manners, ,
1933: [04.28] Upperworld (Cast: Marcus), Directed by Roy del Ruth, with , Mary Astor, Ginger Rogers,
1933: [03.18] Grand Slam (Cast: Cedric Van Dorn), Directed by Wilhelm Dieterle, with Paul Lukas, Loretta Young, Frank McHugh,
1932: [11.05] The Mask of Fu-Manchu (Cast: British Museum Official (/xx/) ), Directed by Charles Brabin, Charles Vidor, with Boris Karloff, Karen Morley, Myrna Loy,
1932: [04.12] Grand Hotel (Cast: Pimenov), Directed by Edmund Goulding, with Greta Garbo, John Barrymore, Joan Crawford,
1932: [02.10] The Sign of the Cross (Cast: Glabrio ), Directed by Cecil B. DeMille, with Fredric March, Elissa Landi, Claudette Colbert,
1931: [12.17] Tonight or Never (Cast: Rudig), Directed by Mervyn LeRoy, with Gloria Swanson, Melvyn Douglas, Alison Skipworth,
1923: [10.21] Zaza (Cast: Duc de Brissac), Directed by Allan Dwan, with Gloria Swanson, H.B. Warner,
1919: [05.31] Broken Blossoms (The yellow man and the Girl) (Composer), Directed by D.W. Griffith, with Lillian Gish, Richard Barthelmess, Donald Crisp,
1918: [02.18] My Wife (Cast: Biggy Gore), Directed by Dell Henderson, with Anna Murdock, Rex McDougall, Hubert Druce,
1917: [11.19] Please Help Emily (Cast: Herbert Threadgold ), Directed by Dell Henderson, with Anna Murdock, Hubert Druce, Amy Veness,
Filmography Ferdinand Gottschalk nach KategorienMusik Darsteller
DisclaimerThe information above does not purport to be exhaustive, but give the level of information to our database at the time of last update. All figures are best knowledge and belief researched to, legal claims arising from incorrect or missing information are expressly rejected.
Due to the current law in some countries can be held responsible person who addresses refers to having a criminal content spread may or criminal content and pages further refer, unless he closes clear from such liability. Our links below are researched to the best of my knowledge and belief, but we can not accept in any way for the content of these web pages and links from us any liability. We provide this explanation by analogy from all countries and all languages of. Quotations, if not otherwise marked, in the usual right to quote with identification of the sources added.
The copyright lies with the authors of these quotes - Ferdinand Gottschalk - KinoTV


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Napa Wines Need Your Help
Yes, Napa needs your support now more than ever. But if the great wine shortage of 2014 comes to pass, buy from these emerging wine regions.
Climate Tourism Is Really Heating Up
Climate change is opening up more than ice fields—it's stoking a new tourism economy.
Best Travel Photography: June 2014
These images, from some of Outside's favorite photographers, will make you want to pack your bags and get lost in adventure.
Escaping the Recession by Boat
When the Great Recession hit, young people found a million different ways to cope with their battered job prospects. Alex and Nick Kleeman found the best way, scraping together enough cash to buy a 32-foot sailboat, then plunging into the Pacific for the adventure of their lives. So what if they didn't know how to sail?
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ComP is a sensor histidine kinase of Bacillus subtilisrequired for the signal transduction pathway that initiates the development of competence for genetic transformation. It is believed that ComP senses the presence of ComX, a modified extracellular peptide pheromone, and donates a phosphate to ComA, thereby activating this transcription factor for binding to the srfA promoter. In the present study, fusions to the Escherichia coli proteins PhoA and LacZ and analysis of its susceptibility to the protease kallikrein were used to probe the membrane topology of ComP. These data suggest that ComP contains six or eight membrane-spanning segments and two large extracytoplasmic loops in its N-terminal membrane-associated domain. Deletions were introduced involving the large extracellular loops to explore the role of the N-terminal domain of ComP in signal transduction. The absence of the second loop conferred a phenotype in which ComP was active in the absence of ComX. The implications of these data are discussed.
Competence development inBacillus subtilis is controlled by a complex signal transduction pathway that culminates with the activation of genes encoding the machinery for DNA binding and uptake (for reviews, see references 11, 12, and 16).
The histidine-kinase ComP (64) and its cognate response regulator ComA (63), members of the family of two-component regulatory proteins (45), are required for competence. ComP is a membrane-bound protein with a C-terminal domain highly conserved among histidine kinases, whereas ComA is a cytoplasmic protein. It is likely, by analogy with other two-component regulatory systems, that ComP autophosphorylates a conserved histidine and subsequently donates its phosphoryl group to the cognate response regulator ComA (64). For competence development, ComA-PO4 must bind to the promoter region of srfA (43, 44, 50) thereby activating its transcription. Embedded in the srfAoperon is comS, a small open reading frame required to activate ComK, the competence transcription factor (7, 17, 59, 60).
Two B. subtilis extracellular peptide factors accumulate in the medium as cells grow to high density and act via converging signal transduction pathways to activate the transcription of srfA(35, 55). ComX, a modified 9- to 10-amino-acid peptide (35) acts via ComP, presumably to increase the phosphorylation of ComA. The response to the other competence pheromone CSF (competence and sporulation stimulating factor) does not require ComP but also depends on ComA (55). CSF, imported by an oligopeptide permease (48, 56), inhibits a phosphoprotein phosphatase which otherwise dephosphorylates ComA (29, 56). Both pheromone pathways therefore regulate the level of ComA-PO4 and consequently determine the rate ofsrfA transcription.
ComP and ComA play a more general role than merely to regulate competence. In addition to srfA, several other loci are dependent on these proteins for their growth-stage-dependent induction. For instance, degQ (41) and several phosphatases which act to dephosphorylate response regulator proteins (including ComA) (42, 47) are regulated by this two-component system. ComP and ComA are therefore important players in the adaptation ofB. subtilis to conditions of high population density.
The amino acid sequence of the large hydrophobic N-terminal domain of ComP does not resemble that of any known protein, whereas the C-terminal moiety of ComP shares similarity with the transmitter domain of other sensor kinase proteins (64). Moreover, this domain has been predicted to contain multiple membrane-spanning segments (64), a topological feature different from most membrane-localized sensors which possess only two transmembrane segments (45, 57).
Since ComX is an extracellular pheromone, it is likely that the membrane domain of ComP plays an important role in ComX detection. In this study we have examined the membrane topology of ComP and tested the functional relevance of portions of ComP predicted to be extracellular, thereby laying the basis for future mechanistic studies. We demonstrate that removal of one of the extracellular loops of the ComP membrane domain renders its activity independent of ComX.
Bacterial strains and growth media.The plasmids andB. subtilis strains used are described in Table1. Escherichia coli strains are derivatives of DH5α and JM109 carrying plasmids pUCCMPHOA and pJF751, respectively (see below). E. coli was grown in Luria-Bertani medium (52) with ampicillin (Sigma) (100 μg/ml). B. subtilis strains were derivatives of strain 168 and were isogenic with IS75 (hisA1 leu-8 metB5). B. subtilis strains harboring fusions were obtained by transformation of IS75 with derivatives of pUCCMPHOA or pJF751 (carryingcomP-phoA or comP-lacZ, respectively) by using selection for chloramphenicol (5 μg/ml; Sigma). The resulting transformants were derived from single crossover events at thecomP locus and carry the comP fusions in single copy on the chromosome downstream of the endogenous regulatory sequences. Transformation of B. subtilis was carried out as described previously (1). For the assay of enzymatic activities, B. subtilis was grown at 37°C in competence medium (1) containing chloramphenicol (5 μg/ml). ThecomX comP::spc deletion strain was constructed by a double-crossover event and replaced all ofcomP and the last four codons of comX by a spectinomycin resistance cassette.
DNA manipulations.Standard procedures were used to prepare and handle recombinant DNA, for the dideoxy sequencing, and to transform the E. coli cells (2). PCRs were carried out with Vent DNA polymerase (N. E. Biolabs) or with AmpliTaq DNA polymerase (Perkin-Elmer).
Construction of comP-phoA and comP-lacZfusions.Fusions of comP to either phoA orlacZ were generated by cloning fragments of comP, amplified by PCR, in frame with the phoA reporter gene of plasmid pUCCMPHOA or with the lacZ reporter gene of plasmid pJF751 (13). Plasmid pUCCMPHOA was constructed by cloning aPstI fragment carrying the E. coli phoA gene, lacking its promoter and the first 14 codons of its sequence, into thePstI site of the pUCCM18 vector (21). Hybrid genes coding for different fusions of the ComP amino-terminal region to the 15th residue of PhoA were constructed by designing PCR primers that placed sections of ComP in frame with PhoA. The upstream primer contained a KpnI site, and the downstream primers each contained SalI sites, which were used for cloning. As a result, the fragments of comP (Table 1) from the start codon to the most distal comP codons (located between D106 and L500) were separated from phoA by four new codons introduced by cloning. These encoded the amino acid sequence SRPA, joined to the first residue (A15) of the PhoA moiety. Similarly, constructs containing several of the same fragments of comP fused to the eight codon of lacZ were generated with primers containing EcoRI and BamHI sites that were then used for cloning. The lacZ fusions each contained codons for the amino acids AD, introduced by cloning, preceding the first residue (P8) of the LacZ moiety. Restriction analysis and DNA sequencing verified the junction of each fusion construct. In all constructs the hybrid genes were located downstream from the lacZ promoter, which was used to drive expression in E. coli. In B. subtilis the fusion constructs were chromosomally integrated at the comP locus and transcribed from the comPpromoter (see above).
Construction of B. subtilis strains expressing mutant ComP proteins.Plasmids allowing inducible expression at theamy locus of ComP derivatives lacking loops a, b, and c (Fig. 1) were generated as follows. A PCR fragment carrying the 545 C-terminal codons of comP (starting from the F225 codon) and flanked by XbaI and ClaI restriction sites, was cloned into pDR67 (22), giving pDR67-′comP. Then, four fragments, flanked by SmaI and XbaI restriction sites and carrying 42, 93, 167 or 224 N-terminal codons ofcomP were obtained by PCR and cloned into pDR67-′comP to give, respectively, pDR67-ΔLabc-comP, pDR67-ΔLbc-comP, pDR67-ΔLc-comP, and pDR67-Xba-comP plasmids. The pDR67-ΔLabc-comP, pDR67-ΔLbc-comP, and pDR67-ΔLc-comP plasmids contain the sequences encoding ComP derivative proteins missing loops a, b, and c (ΔLabc-ComP), loops b and c (ΔLbc-ComP), or only loop c (ΔLc-ComP). The pDR67-Xba-comP plasmid encodes a ComP protein, derived from the wild type by insertion of S and R residues, in order to introduceXbaI termini for ligation. This construct serves as a control for the others, which also contain an XbaI site at the same position. All of the cloned fragments derived by PCR were completely verified by sequencing. All of these plasmids were used to transform IS75, and chloramphenicol-resistant transformants that had integrated the mutant comP genes at the amyElocus were identified. The resulting strains were then transformed by congression with chromosomal DNA prepared from BD1890 (srfA-lacZ) together with DNA from either BD1853 (comPΔBclI) or BD2356 (comXP::spc), in order to introduce asrfA-lacZ transcriptional fusion while simultaneously inactivating either the comP gene alone or both thecomX and comP genes.
Assay of alkaline phosphatase and β-galactosidase activities.Alkaline phosphatase activity in E. coli, conferred by the comP-phoA fusions, was determined in liquid cultures by using p-nitrophenyl phosphate (Sigma) as described previously (36). The same assay was used forB. subtilis cultures grown in competence medium toT0, except that cell permeabilization with detergent and chloroform was omitted and the B. subtiliscells were concentrated fivefold by centrifugation prior to assay. InB. subtilis, alkaline phosphatase activity values were corrected for the low background level exhibited by the control strain IS75. DH5α was used as the host for alkaline phosphatase assays inE. coli, since we detected no background alkaline phosphatase activity in this strain. The alkaline phosphatase activity determinations presented in this study were performed on aliquots of the same samples that were worked up for Western blot analysis. For assay of the comP-lacZ fusion strains, the β-galactosidase specific activity was determined in B. subtilis and E. coli as described earlier (1, 40). The E. coli host strain used for the β-galactosidase assays was JM109. For assay of srfA-lacZ expression in the comPdeletion mutants of B. subtilis, strains were grown in competence medium with or without IPTG (isopropyl-β-d-thiogalactopyranoside; 1 mM), and 500-μl samples were collected every hour. β-Galactosidase activities were measured in the kinetic mode on a Spectra Rainbow (Tecan) microplate reader. For the calculation of specific activity, the total protein was estimated from Klett readings on culture samples by using an experimentally determined calibration curve.
Preparation of protoplasts and right-side-out membrane vesicles from B. subtilis.Protoplasts were prepared as described previously (6), and protoplast formation was monitored by microscopic examination. For the preparation of membrane vesicles, hypotonically lysed protoplasts were incubated in the presence of DNase I (10 μg/ml; Sigma) at room temperature for 10 min, and the Mg2+ concentration was subsequently adjusted to 5 mM. Membranes were purified by isopycnic centrifugation through a discontinuous sucrose gradient as described earlier (34, 58), washed in 0.1 M sodium phosphate buffer (pH 7.2)–1 mM phenylmethylsulfonyl fluoride, and stored at −20°C in the same buffer.
Proteolysis with kallikrein. B. subtilis protoplasts were resuspended in 33 mM Tris-HCl (pH 8.0)–100 mM KCl–5 mM MgSO4–0.5 M sucrose at a protein concentration of 0.5 mg/ml. Triton X-100 (0.5%) was added to some of the samples as noted. Samples (0.5 mg/ml) were incubated with kallikrein (Sigma) at a final concentration of 100 or 200 μg/ml. In some cases Triton X-100 (0.5%) was added to permeabilize the protoplasts. Proteolysis was carried out by incubation for 3 h at 37°C. The protease inhibitor leupeptin (1 μM; Sigma) was added to stop the reaction. Samples were centrifuged, and the pellet (membrane fraction) was resuspended in sample buffer (28). From each sample, 30 μg of protein per lane was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% gel. The proteins were transferred to nitrocellulose and analyzed by immunoblotting with anti-ComP antisera.
SDS-PAGE and immunoblotting.Proteins from B. subtilis membrane preparations or from E. coliwhole-cell extracts were resolved on 10% gels by SDS-PAGE (28) without previous boiling of the samples. Gels were equilibrated in transfer buffer (48 mM Tris-HCl, pH 9.2; 29 mM glycine; 0.05% SDS; 20% methanol) for 10 to 15 min and then electrophoretically transferred to pre-wetted nitrocellulose membranes (0.45-μm pore size; Schleicher & Schuell) for 15 min at 15 V in a semidry transfer apparatus (Bio-Rad). Nitrocellulose membranes were incubated with monoclonal anti-PhoA antibody (Boehringer) or with a guinea pig anti-ComP antiserum directed against a synthetic peptide corresponding to the last 16 aminoacyl residues of ComP and then with a peroxidase-conjugated secondary antibody, which was detected by using a Chemiluminescent Substrate Kit (Kirkegaard & Perry Laboratories) as recommended by the supplier. The protein concentration was estimated by using the Bio-Rad protein assay reagent.
Based on hydropathy analysis of ComP, eight hydrophobic segments were predicted to be membrane spanning (64), leaving two large regions each consisting of approximately 80 amino acids outside the cell membrane and the C-terminal histidine kinase domain in the cytoplasm (Fig. 1). We have used Western blotting on fractionated lysates to confirm that ComP is localized exclusively in the cell membrane of B. subtilis and behaves as an integral membrane protein with respect to NaOH solubility (not shown) (51).
Deletion of the large external loops of ComP relieves it from ComX control.Based on the hypothetical topology displayed in Fig. 1, we tested the involvement of the most N-terminal loops (a, b, and c) of ComP in the ComX quorum sensing pathway (see Fig. 1 for the identification of these loops). The expression of asrfA-lacZ transcriptional fusion was measured as an indication of activation of the quorum sensing pathway. We constructed three isogenic B. subtilis strains, all with an inactivating deletion of comP and expressing, from a pSpac (IPTG-inducible) promoter, three ComP derivative proteins with deletions of either loop c (in BD2759), loops b and c (in BD2758), or loops a, b, and c (in BD2757). These constructions were designed so that 13 residues remained between the C terminus of the deleted segment and the N terminus of the following transmembrane segment. These residues were intended to serve as an extracytoplasmic linker connecting the up- and downstream portions of the protein in order to minimize the disruption of the topology of the mutant ComP proteins. The constructions introduced anXbaI restriction site, adding S and R residues after position 224 in all the ComP derivative proteins. We also constructed the control strain BD2760, which expressed an otherwise wild-type ComP but contained the S and R insertions.
These strains were grown in competence medium with or without 1 mM IPTG, and their β-galactosidase activities were measured during growth (Fig. 2). Little or no β-galactosidase activity was detected after growth in the absence of IPTG, as expected. When expression of the comP derivative genes was induced by the addition of IPTG, srfA-lacZ was expressed in all the strains at levels comparable to that in the wild-type strain (BD1890). Thus, all of these mutant ComP proteins can replace wild-type ComP for the induction of srfA expression in vivo.
To determine the dependence of the activities associated with the mutant ComP proteins on ComX, we performed the same experiment with these comP derivatives in a comX comP background. Very little activity was detected in the absence of IPTG (Fig. 2). The control strain (BD2747), expressing ComP with the S and R insertions in the comX comP background, did not express detectable β-galactosidase activity even when grown in the presence of IPTG (Fig. 2D). This phenotype could be fully complemented by growth in ComX-containing conditioned medium prepared from the wild-type strain BD1890 (Fig. 3), demonstrating that ComP with the S and R insertions responded to the ComX pheromone, as well as the wild-type protein. However, all strains carrying deletions in the N-terminal loops no longer required ComX forsrfA-lacZ expression (Fig. 2). comX inactivation did not affect the activities of the ComP derivative proteins lacking either loops b and c (in BD2745) or loops a, b, and c (in BD2744) (Fig.2A and B). BD2746, expressing a ComP protein lacking only loop c, exhibited approximately a twofold-lower level of srfA-lacZactivity in the absence of endogenous ComX than did the other deletion strains (Fig. 2C). This was observed in three independent experiments. In spite of this difference, the loop c deletion strain did not respond to conditioned medium (Fig. 3).
Based on the topological model shown in Fig. 1, we may conclude that deletion of loop c relieved the protein from ComX control, at least partially, while deletion of loops b and c completely eliminated ComX dependence. Interpretation of the deletion results obviously depends on the topology of the three most N-terminal loops. However, computer modeling cannot be regarded as definitive. For example we cannot be confident that loops a and c are extracellular, since the polarity of the proposed structure was based solely on the distribution of positive charges (61, 62), and this portion of the structure might actually be reversed. Therefore, we have tested the model by constructing in-frame fusions of comP to a deleted version of the E. coli phoA gene, encoding mature alkaline phosphatase.
Analysis of ComP-PhoA fusions: topology of loops a, b, and c.Alkaline phosphatase has been used as a reporter for the subcellular localization of different portions of a protein (reviewed in references37 and 38) by exploiting the fact that it is active only when translocated across the membrane (8). Eleven 3′-deleted fragments of comP were cloned in frame with phoA in the pUCCMPHOA vector. Nearly all of the fusions contained junction points at the C termini of each of the predicted hydrophilic regions of ComP. This strategy was intended to minimize the disruption of topological signals (e.g., membrane-spanning segments and hydrophilic regions with positive net charges) (5). Fusion points are listed in Table2, and their positions are indicated schematically in Fig. 1. Since the principal aim of these experiments was to test the topological model for the predicted loops a, b, and c, we will first discuss the fusions that are relevant to this purpose.
The recombinant phoA fusion plasmids were tested in B. subtilis after chromosomal integration in single copy. Alkaline phosphatase activity was measured in cells grown in competence medium (1). Expression of the endogenous B. subtilisvegetative alkaline phosphatase was repressed by the high concentration of phosphate in this medium (20). The alkaline phosphatase activities (Table 2) were normalized to the highest activity (fusion H). Fusions H, F, and L were associated with high activity and fusion I failed to exhibit any detectable activity above the background. Western blot analysis of purified membrane vesicles from B. subtiliswas carried out on these fusion strains by using anti-PhoA antibody. All the fusion proteins were detected in the cell membrane fraction (Fig. 4). A product with an electrophoretic mobility similar to that of mature alkaline phosphatase, presumably formed by degradation, was detected for some fusions and no signal was detectable in the control strain with no fusion (Fig. 4, lane 10). Strong signals were associated with fusions H, I, and M, and weak signals were associated with fusions F and L. It is noteworthy that, with the exception of H, the fusions that place alkaline phosphatase outside the membrane exhibited relatively high alkaline phosphatase activity but weak Western blot signals. Possibly, PhoA tends to be unstable in B. subtilis either in vivo or in the extracts, when exposed on the exterior face of the membrane. However, since low alkaline phosphatase activity was not associated with weak Western blot signals, the B. subtilis results provide strong support for the topological model concerning loops a, b, and c and buttress our interpretation of the structures affected by our deletion mutations.
The recombinant phoA fusion plasmids were also tested inE. coli DH5α because the relative paucity of published reports on the use of PhoA fusions in B. subtilis made it advisable to compare results obtained in the two organisms. Alkaline phosphatase activities obtained in E. coli, normalized to that of fusion H which exhibited the highest activity, are shown in Table 2. Fusion proteins H and F exhibited high alkaline phosphatase activity, suggesting that residues D106 and R112 of ComP (Fig. 1 and Table 2) were extracytoplasmic. Fusion L was associated with low but measurable activity, suggesting that residue E223 was possibly extracytoplasmic as well. In contrast, no detectable alkaline phosphatase activity was exhibited by fusions I and M. Residues R143 and K272 were therefore judged to be cytoplasmic. Since the alkaline phosphatase activity will depend on the abundance of each fusion protein in addition to the location of the PhoA moiety, PhoA antiserum was used to detect the fusion proteins in Western blot analysis (Fig.5). Strong signals were detected for fusions I, F and H. The abundance of the fusion I protein, together with the absence of detectable activity, provided strong support for the cytoplasmic location of R143. Fusion L was evidently much less abundant than F and H, probably due to lower stability. This provided a reasonable explanation for the relatively low activity exhibited by fusion L. Fusion M was at least as abundant as several others that were associated with readily detectable alkaline phosphatase activities. Taken together, the phoA fusion results from E. coli strongly support the predicted ComP topology for loops a, b, and c, and are consistent with the results obtained in B. subtilis.
Analysis of ComP-PhoA fusions: topology of the distal portions of the ComP N-terminal domain.PhoA fusions were also used to test the more distal portion of the model, beyond fusion point M (Fig. 1 and Table 2). In both B. subtilis and E. coli, fusion P (I362) exhibited relatively high activity and fusion Z (P314) was associated with low but detectable activity. No activity was detected in the cases of fusions O (Y338) and Y (L500). The behavior of these fusions was consistent with the model. However, the absence of activity with fusion N and the activity associated with fusion G do not support the model presented in Fig. 1. The C terminus of ComP is predicted to be located in the cytoplasm since its histidine kinase domain must be accessible to ATP and to the response regulator ComA. The behavior of fusion G was at variance with this expectation, since it exhibited a higher activity than other fusions with junctions located in the cytoplasm. This discrepancy may be due to the absence in fusion G of an important determinant of cytoplasmic localization. Fusion Y (L500) exhibited no detectable AP activity, suggesting that this putative topogenic signal is located between the junctions of fusions G and Y. In fact, between positions 383 and 400 there are six positively charged residues, not included in fusion G, which may comprise such a cytoplasmic determinant (61, 62). The behavior of fusion N is more difficult to interpret. The absence of fusion N-associated activity was not explicable by a low level of synthesis or the instability of the fusion protein. Western blots revealed a clear signal for the fusion N protein, one considerably stronger than that of fusion P, which had a relatively high AP activity (Fig. 4, 5, and6). The weak Western blot signals for fusion P in both hosts (and for F and L in B. subtilis) indicate either protein instability in vivo, associated with high specific activities, or more likely instability in extracts.
Analysis of ComP-LacZ fusions.The data obtained with the ComP-PhoA fusions described above were generally in agreement with the topology proposed for ComP, especially for the most relevant region encompassing loops a, b, and c. However, the behavior of fusion N in particular failed to support the model. An alternative would hold that the region including membrane-spanning segments 5 and 6, between K272 (fusion M junction) and Y338 (fusion O junction), is located in the cytoplasm.
To further test the model, we constructed comP fusions tolacZ and determined their β-galactosidase activities inE. coli and B. subtilis. When LacZ is used as a reporter protein for membrane topology, a reversal of relative enzymatic activities compared to that of PhoA fusions is observed, since fusion proteins between the amino-terminal segments of membrane proteins and LacZ are active enzymatically only when the LacZ moiety of the hybrid is localized in the cytoplasm (15, 31, 54). ThecomP-lacZ gene fusions were generated with a strategy similar to that used for cloning the comP-phoA fusions (see Materials and Methods). Plasmid derivatives of JF751 carrying fusions to lacZ are listed in Table 1. The seven fusion points, indicated in Table 2 and Fig. 1, were identical to those in the corresponding PhoA fusions.
In B. subtilis, the LacZ constructs were again inserted chromosomally in single copy. The ambiguity with the phoAfusion N was reflected with fusion S. Although lower than the activities of fusion R, T, and X, which clearly place thelacZ moiety in the cytoplasm, fusion S displays a higher activity than the extracellular fusion U. The low activity of fusion V may once more be explained by the argument that this fusion, as in the case of the corresponding fusion to PhoA (fusion G), excluded important downstream topological determinants for cytoplasmic localization. In summary, the lacZ fusion experiments did not lift the uncertainty associated with segments 5 and 6, and so we cannot clearly exclude the possibility that these segments are located in the cytoplasm.
E. coli strains carrying the comP-lacZ gene fusions were also tested for β-galactosidase activity (Table 2). The activities associated with fusions R, T, and X, predicted to place the LacZ moiety in the cytoplasm, are generally higher than those of fusions S, W, and U, which should place LacZ outside the membrane. These data support the original model.
Protease susceptibility of ComP.Additional evidence that the amino-terminal part of ComP contains regions exposed on the outer surface of the membrane was obtained from protease susceptibility studies with protoplasts. We used the protease kallikrein, which cleaves the peptide bond N-terminal to FK and LR sequences. ComP contains six potential cleavage sites for kallikrein (Fig. 1). Of these, three are predicted to be extracytoplasmic: two in loop a and one in loop c. Only these three sites will be accessible to kallikrein in intact protoplasts. Our ComP antiserum was raised against a synthetic peptide corresponding to the ultimate 16 C-terminal residues. The respective sizes of the C-terminal proteolytic products generated from cleavage at each of the extracytoplasmic sites would be 79.9, 76.0, and 67.3 kDa. In a limit digest only the 67.3-kDa product would be detectable.
Protoplasts from a B. subtilis strain (BD2358) that expresses ComP from a multicopy plasmid were treated with kallikrein for 3 h, and membrane preparations were analyzed by Western blotting (Fig. 6). The integrity of the protoplasts was confirmed in Western blots by the resistance to kallikrein of the cytoplasmic MecA protein (26), which contains a single, centrally located cleavage site. A prominent proteolytic product of approximately 65 kDa was detected when protoplasts from the strain expressing ComP were treated with kallikrein (Fig. 6, lanes 5 and 6). The 65-kDa signal was absent in kallikrein-treated protoplasts from a comPdeletion strain (BD2356), confirming that it was derived from ComP (Fig. 6, lane 1). This product was also absent from extracts prepared from protoplasts incubated without kallikrein (Fig. 6, lanes 7 and 8) and from protoplasts treated with Triton X-100 together with kallikrein (Fig. 6, lanes 3 and 4). In the presence of Triton X-100, protoplasts are permeabilized and cytoplasmic cleavage sites become accessible to the protease, as indicated by the degradation of MecA (data not shown). The ComP-derived products generated by cleavage at these sites are too small to be visible in the Western blot shown in Fig. 6. A strain with a total ComP deletion (BD2356) was included as a negative control (Fig.6, lanes 1 and 2). In extracts from this strain a proteolytic product with a size similar to that of ComP was detected, presumably originating from a protein that cross-reacts with the antiserum. This product obscured the disappearance of ComP in lanes 3 to 6.
The ComP-specific kallikrein degradation product with a nominal molecular size of 65 kDa probably corresponds to the predicted 67.3-kDa product. This is the expected product from a limit digest of the three sites predicted to be extracellular. The presence of a unique ComP-derived cleavage product obtained only when intact protoplasts were treated with kallikrein supported the conclusion that a hydrophilic portion of the N-terminal domain of ComP, most probably corresponding to loop c, is extracytoplasmic, lending further support to our topological model.
The phoA and lacZ fusion experiments established that ComP is a polytopic integral membrane protein with two large extracellular segments. The kallikrein accessibility experiment supported these conclusions since it was consistent with extracellular exposure of a portion of the N-terminal domain. The cytoplasmic location assigned to the C-terminal hydrophilic domain is in accord with the putative role of ComP as an autokinase.
There have been relatively few examples of the use of protein fusions to determine membrane protein topology in gram-positive organisms (14, 21). The normalized activities of the phoAand lacZ fusions are similar in B. subtilis and in E. coli, suggesting that in B. subtilis, which lacks a periplasm in the strict sense (but see reference39), fusions to phoA and lacZgive results similar to those obtained in E. coli.
The greatest uncertainty in our data concerns the location of the N/S fusion junction, raising the possibility that the entire region, which includes the predicted fifth and sixth membrane-spanning segments, is actually cytoplasmic. Contrary to our original prediction, no detectable alkaline phosphatase activity was associated with fusion N. Also, the normalized β-galactosidase activity associated with fusion S in B. subtilis does not unambiguously support either model. However, the possible presence of eight membrane-spanning segments could be rationalized as follows. The alkaline phosphatase activities of fusions N, Z, and P increase with the distance of the fusion junctions from the N terminus of ComP (Table 2). This pattern is consistent with the possibility that the correct membrane insertion of segments 5 and 6 requires the interactions of protein sequences located downstream from the S/N fusion point (Y299) and that the deletion of these sequences results in decreased probabilities that the correct insertion will occur. Perhaps a domain, which includes transmembrane segments 6 to 8 must fold properly before the region from 5 to 6 can undergo membrane insertion. An alternative possibility is that segments 5 and 6 (Fig. 1) are embedded in the membrane but do not completely cross it. In summary, we conclude that ComP contains either six or eight membrane-spanning segments.
The most important conclusion from the topology studies is that it confirms the extracellular locations of loops a and c and the cytoplasmic disposition of loop b. Removal of the extracellular loop c or of a segment including loops b and c or loops a, b, and c confers a striking phenotype, rendering srfA-lacZ expression independent of ComX. These data imply that sequences included within loop c are required to maintain ComP in an inactive state for autophosphorylation or to activate a dephosphorylation activity and that ComX may serve to antagonize this inhibitory state. In the mutant proteins in which loop c is deleted, this state cannot be established and ComP is active and presumably phosphorylated, even in the absence of ComX. The lower srfA-lacZ activity of the ΔLc strain compared to that of the ΔLabc and ΔLbc strains (Fig. 2) was observed reproducibly in the comX background. It may be that removal of just loop c is not sufficient for full srfAexpression in the absence of ComX. However, the ΔLc strain does not respond to extracellular ComX by increasing the level ofsrfA-lacZ expression (Fig. 3). This suggests that loop c is essential for the signal transduction mechanism that detects the presence of ComX (directly or indirectly). If ComP interacts directly with ComX, then loop c may be essential for this binding. Our data in no way rules out the possibility that residues from loops a and b are also required for interaction with ComX.
In additional experiments (50a), three deletion mutations were constructed that lacked the entire N-terminal hydrophobic domain of ComP. The deleted genes were integrated in a single copy in theB. subtilis chromosome, and the activities of the cognate proteins were determined by using a srfA-lacZ reporter in acomP background. Very low or no β-galactosidase activity was detected with the deletions. When these deletion proteins and a wild-type ComP were expressed from identical plasmids in B. subtilis, Western blotting revealed comparable cellular levels of the truncated and wild-type proteins. Removal of the membrane-localized N-terminal domain of ComP resulted in a dramatic decrease in activity, and the deleted constructs could not respond to ComX when tested with conditioned medium. This suggests that the portions of the N-terminal domain downstream from loops a, b, and c are required for the activity of ComP. We propose therefore that loops a, b, and c (notably loop c) play a role in response to ComX, whereas the downstream region comprising membrane-spanning segments 5 to 8 is needed for ComP function, either as an autokinase or to suppress a phosphatase activity.
It is notable that the growth stage regulation of srfA-lacZexpression in the strains carrying the ComX-independent forms of ComP is not altered; induction of srfA-lacZ expression begins atT0 in these strains as it does in the wild type (Fig. 2). This indicates that the mechanism of growth-stage-dependent control of srfA-lacZ expression is redundant and does not depend exclusively on the ComX signaling pathway. Growth stage-specific expression of srfA is known to be regulated negatively by CodY (53) and positively by CSF (30) and SinR (33).
Most membrane-localized histidine kinases possess two membrane-spanning segments and a single extracytoplasmic loop. ComP is a member of a growing family of membrane-localized bacterial histidine kinases that possess more-complex structures containing several membrane-spanning segments. Most of these proteins are involved in quorum-sensing signal transduction mechanisms that use peptide pheromones, often with posttranslational modifications. For instance, a quorum-sensing system, analogous to the ComP-ComA two-component signal transduction pathway, regulates competence in S. pneumoniae (18, 49). In this system a peptide pheromone interacts with the histidine kinase protein ComD (19). ComD is predicted by hydropathy analysis to contain five to seven transmembrane segments (19) but displays no obvious sequence similarity to ComP except in the cytoplasmic histidine kinase domain. The quorum-sensing histidine kinase AgrC, which regulates virulence in S. aureus in response to a peptide pheromone (25, 27), also contains multiple membrane-spanning segments (32). The synthesis of antibiotics in Lactobacillus and Carnobacteriumspp. is regulated by the histidine kinases PlnB (9, 10), SapK (3, 4), and SppK (3, 4) that are related to ComD (19) and also appear to sense the presence of peptide pheromones. These kinases possess hydrophobic N-terminal domains, each about 200 residues long, predicted by hydropathy analysis to contain five to eight membrane-spanning segments (4, 10, 19).
Although ComD and AgrC appear to detect their cognate peptide pheromones directly (19, 24), this has not been demonstrated in the cases of other quorum-sensing histidine kinases, including ComP. The similarities in their membrane topologies noted above suggest that signal transduction in these various systems may have common features. Further insight into the mechanism of ComX-ComP interaction may be obtained by the genetic and biochemical analysis of additional ComP mutations specifically affecting the response to ComX. Localization of these mutations with respect to the membrane topology described in this study would help to identify the determinants involved in interaction with ComX and in the response to this interaction.
We thank the members of our laboratory, especially G. Inamine, for useful discussions and advice. We also thank Eugenio Ferrari for contributing JF751 and Manuella Roggiani for constructing threecomP deletions.
This work was supported by NIH grant GM57720 and by a Lavoisier Fellowship to P.T. awarded by the French Ministry of Foreign Affairs.
- Received 15 March 1999.
- Accepted 17 May 1999.
- Copyright © 1999 American Society for Microbiology


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Doubly disadvantaged : gender, spatially concentrated deprivation and nascent entrepreneurial activity
Murzacheva, Ekaterina and Sahasranamam, Sreevas and Levie, Jonathan (2019) Doubly disadvantaged : gender, spatially concentrated deprivation and nascent entrepreneurial activity. European Management Review. ISSN 1740-4754 (In Press)
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Drawing on human capital, intersectionality and mixed embeddedness theory, we test hypotheses on the relationship between gender differences in human capital and gender differences in nascent entrepreneurial activity across geographical space, and the moderating effect of spatially concentrated deprivation on this relationship. Using UK data from Global Entrepreneurship Monitor, we find that the disadvantaged position of female nascent entrepreneurs arises from social exclusion, and specifically that the gender differences in nascent entrepreneurial activity are directly related to differences in general and specific human capital across locales. Moreover, in deprived locations, women as a group do not gain from any human capital advantage they might have over men, causing a double disadvantage for women. Our results make a novel contribution to the literature on disadvantage entrepreneurship, and we discuss policy options to tackle double disadvantage in deprived locales.
|Murzacheva, Ekaterina, Sahasranamam, Sreevas ORCID: https://orcid.org/0000-0001-9418-4493 and Levie, Jonathan;
|Item type:
|ID code:
|entrepreneurship, gender, multiple deprivation, global entrepreneurship monitor, human capital, inequality, female entrepreneurship, disadvantaged communities, Commerce, Economics, Econometrics and Finance(all)
|Social Sciences > Commerce
|Strathclyde Business School > Hunter Centre For Entrepreneurship
|Depositing user:
|Pure Administrator
|Date deposited:
|15 Oct 2019 14:42
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|28 Jun 2020 01:47
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Eleanor Ridley
What did you do prior to starting at Heriot-Watt?
I completed my BSc in Geology & Petroleum Geology at the University of Aberdeen. I also worked part-time at a small oil company: Challenger Minerals (a subsidiary of Transocean) during the final two years of my undergraduate degree.
What have you done since graduating from Heriot-Watt?
After three months of job searching, I joined Challenge Energy as a Geoscientist. Challenge Energy is a small oil and gas consultancy based in Guildford, England.
What has been the most useful or enjoyable aspect of your course?
The field trips were really enjoyable. Another bonus has been the continued contacts and good friends now in the industry. The most useful parts were the subjects covered, but also the use of up-to-date software, which is extremely useful for a career in the industry.
How has the course helped your career and professional development?
The course qualification enabled me to enter the competition with all the other graduates in the oil and gas industry. Without an MSc it is very difficult to even be in the running, and it is highly unlikely to be successful at job applications without it. The course material provided has also been fantastic for referencing whilst at work.
The department staff were always very welcoming and supportive if ever you needed help – even if it was on a Sunday!


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Client: Stevens Group
Architect: EJE Architecture
Delivery Form: Design and Construct
Location: Toronto, NSW
Value: $25.3m
Completion date: September 2020
Foreshore Toronto consists of 37 high end residential units situated on the waters edge of Lake Macquarie. A collection of 1, 2 and 3 bedroom apartments are situated on a 60 meter water frontage, looking over the property's exclusive jetty and 18 swing moorings.
The project includes the redevelopment of the former Hirecraft Marine site, with 100 – 150 square meter apartments each equipped with lift access and basement parking. The addition brings a distinctive contemporary design to Lake Macquarie's stunning waterfront setting.


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Cleveland Clinic
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Prepare for Exam Success with Cleveland Clinic GI Board Review OnDemand
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Cleveland Clinic GI Board Review OnDemand lets you gain an in-depth knowledge of gastrointestinal conditions that are commonly seen in practice, as well as rare but "classic" clinical vignettes that are tested during board-type exams.
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Get On Top Of Google! And Stay There. Being an entrepreneur and running your own business is now more trendy than ever. For those who are new to the game…Read more
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A young couple argue and break up, triggering a chain of events that will transform two families. Donna Vitucci's debut novel tackles a vital human question: How do we prevail when the weight of loss and regret threatens to drag us under? Vitucci's writing is compassionate and wise. At Bobby Trivette's Grave is a story to savor and, finally, be heartened by.
—David Long, author of The Falling Boy and The Inhabited World


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Serous cysts in the abdominal cavity of 6 months old mixed breed pigs
Multiple cysts were observed in the abdominal cavity of two mixed breed 6 months old pigs, with transparent liquid content .
In one of the pigs the cyst was single, located in the mesenterium, with an opaceous wall and serous liquid content. In the other case multiple cysts were found adhered to the spleen capsule, with transparent wall and also serous liquid content.
Morphologically these lesions are consistent with Cysticerculs tenuicollis cysts (Taenia hydatigena larval stage). To confirm this diagnosis the scolex of the parasite should be identified within the cysts. Another possibility, which is more probable in the present case, consists of congenital serous cysts without pathological significance.
To confirm such diagnosis send refrigerated samples (same day) or fixed with formaline (for histopathology) or alcohol for parasitology identification, both fixatives will prevent autholysis.
This entry was posted by SESC on 29/05/2012 at , and is filed under Abdominal cavity (porcine), Lymphoid (porcine), Porcine. Follow any responses to this post through RSS 2.0.You can leave a response or trackback from your own site.
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ENOPION Theatre Company presents The Life & Times of Petunia Butterby
What if all your life you carry with you a memory? A memory of where you were, before you were.In this beautiful telling about a woman named Petunia, you will find the answer to that question.Petunia was born 1869, London England; where she was left on the doorsteps of the Butterby Convent. She was raised by the Nuns there in the only way they knew --- in devotion to God. But there is something different about Petunia, something you will never understand unless you meet her.
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GPSNR Policy Components: Demonstrating a Commitment to Sustainable Natural Rubber
The Member Consultation period for the proposed GPSNR Policy Components and Baseline Reporting Requirements closed on 3 July 2020. All comments received as part of the consultation exercise were discussed by the Policy Toolbox Working Group over several rounds of calls. There has also been healthy dialogue between various membership categories and the Working Group on the alignment of the Policy Components.
Meanwhile, pilot testing for the Baseline Reporting Requirements is in progress, with 10 GPSNR member volunteers participating in the exercise. The Secretariat will be interviewing each of the volunteers in the coming weeks to gather feedback on their experience in submitting the Baseline Reporting Requirements data. More updates will be shared in due course.
At this stage, finalized versions of the Policy Components and related documents have been submitted by the Policy Toolbox Working Group to the Executive Committee for review and further discussion at the Executive Committee level. The Policy Components will then be brought to the GPSNR General Assembly for voting.
The formulation of Policy Components is a critical piece in fulfilling GPSNR's vision of a fair, equitable and environmentally sound natural rubber value chain.
All GPSNR members, at the time of joining the Platform, committed to this very vision, as well as 12 principles of sustainable natural rubber as defined by GPSNR. The Policy Components were developed to provide more specificity to the 12 Sustainable Natural Rubber Principles. Within the proposed Policy Components are key commitment details to no deforestation/ conversion/ degradation, upholding human rights, supply chain transparency and monitoring and reporting.
If approved, the Policy Components will act as a clear and guiding framework for GPSNR company members to establish or update supply chain commitments through their natural rubber purchasing policies.
Adopting a policy allows a company to signal that the issues covered by the policy are important to the company, and that resources and capacities will be dedicated to addressing the issues in order to comply with the policy. Policies, while illuminating the goals that a company is working towards, also enables these commitments to be channelled upstream to suppliers and downstream to buyers, with the potential to transform the entire supply chain.
Following the Policy Toolbox Working Group's observation that common questions and concerns were raised by GPSNR members during the Members Consultation period, the Working Group has developed a supplementary FAQ document to provide detailed responses to address these concerns, in a bid to provide greater clarity on the Policy Components. These will be circulated to the GPSNR membership in August, along with all other proposed resolutions.


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