Objectives Description Deliverables Partner Biodiversity Project: MICRODIV1 Microbial Biodiversity and Biotechnological Opportunities in the Humid Tropics (Microbial Technodiversity) Practical Course Content 1. Discovery of Novel Biotechnological Useful Microorganisms and the Requirement of Identification Today a variety of microorganisms are used in different industrial applications as producers of e.g. antibiotics, enzymes, food colorings, flavorings, organic acids and vitamins. The discovery of a new potential industrial application of a microorganism depends to a large extent upon the discovery of new types of microorganisms and their metabolites. The success of industrial screening programs depends on both the number and the diversity of organisms examined. Many companies therefore are focusing on the isolation and screening of new and rare bacteria and fungi in order to increase the possibilities of discovering new products. The classification and identification of a microorganism is important in the discovery, development and manufacture of a microbial product. A reliable identification improves the effectiveness of a screening program by the elimination of previously studied organisms. The identity of a novel microorganism is also important for safety evaluation and risk assessment of the organism, as well as the approval of the final product. Additionally a characterization of a new isolated microorganism is needed for e.g. patent applications, registration approval, process validation, and quality control respectively. However, the importance to classify and identify microorganisms has often been a neglected task in industrial biotechnology. In this seminar the different taxonomic methods to obtain genotypic and phenotypic information on bacterial and fungal taxa, as well as the requirements for identification in the industrial exploitation of a microbial product, are summarized. 2. Antibiotics: Enrichment and Isolation of Microorganisms Producing Antimicrobial Substances Antibiotics are metabolic products of microorganisms that prevent growth of other microorganisms even in minimal concentrations. Depending on their biological activity, different types of antibiotics, such ß-lactames, macrolides, tetracyclines or peptide-antibiotics can be classified. The primary use of antibiotics is in the treatment of human infectious diseases, and a significant number have agricultural and veterinary uses. Worldwide antibiotic production was estimated at a value of about $16 billion in 1995. Most commercially important antibiotics are produced either by fungi or by bacteria. The overwhelming majority of agents from bacterial sources are produced by Streptomyces spp. or other organisms of the Actinomycetes line and others are the products of various Bacillus species. Table 1 gives some commercially produced antibiotics. 3. Microbial Enzymes: Screening for Microorganisms Capable of Producing Proteases, Lipases, Amylases Cellulases and Arginine Deiminases Todays use of microbial enzymes is mainly focused on production processes in the food industry, on production of washing powders and detergents, and applications in the textile manufacture. Most enzymes are excreted by the microorganisms and can be harvested from the substrate where they are cultivated. Microbial enzymes have many other important applications, notably in organic synthesis, medical diagnostics and research activities. During the workshop the main focus will be on microorganisms capable of producing the following enzymes: Lipases Proteases a-Amylases Ligninases, Cellulases, Hemicellulases Arginine Deiminase Lipases: The food industry exploits lipase-catalyzed reactions to manufacture fats of defined composition and to improove the flavour of food. Numerous microorganisms, such as the bacterium Pseudomonas fluorescens and the fungus Aspergillus niger secrete lipases in their growth medium to catalyze the degradation of lipids. These enzymes can thus be produced on a large scale by fermentations (e.g. the production of cacoa butter uses fungal lipases to convert 1,3-dipalmitoyl-2-oleylglycerol from palm oil). Proteases: Several thousand tons of microbial proteolytic enzymes are produced annually for use as additives to laundry detergents. During washing of clothes, the proteolytic enzymes degrade the proteins present in stains from foodstuffs, blood and so on. About 80% of laundry detergents on the market contain proteolytic enzymes at 0.015 - 0.025% by weight. Because of the special application in laundry processing the proteases used have to be tolerant to a high pH and to high temperatures. ?-Amylases: In textile fabric manufacture, the warp is highly stressed mechanically during weaving. Application of starch paste (sizing), reinforces the warp and prevents loss of string by friction, as well as generation of static electricity on the string. However, for subsequent processing of the cloth (dying, bleaching, finishing) the starch has to be completly removed. Stable bacterial ?-amylases at high temperatures obtained from either Bacillus subtilis or Bacillus licheniformis are used in desizing processes to remove the starch from the textile. Ligninases, Cellulases and Hemicellulases: Decomposition of wood components (lignin, cellulose and hemicellulose) in nature is performed by fungi and bacteria by means of secreted extracellular enzymes that degrade the polymeric compounds and utilize the breakdown products as source of cell carbon and energy. White-rot fungi degrade lignin and polysaccharides at similar rates and prefer hard woods. Brown-rot fungi degrade preferentially polysaccharides and mainly attack soft woods. Bacteria poorly degrade lignins but produce cellulases and hemicellulases capable to degrade polysaccharides. Once the cellulose and hemicellulose is exposed by fungal delignification further degradation is accomplished by fungi and bacteria together. Enzymatic degradation of hemicelluloses is mainly focused on xylans. Paper manufacture generates effluents from wood pulping and pulp processing that contain large amounts of xylans and pollute streams and rivers. Examples of microorganisms that are able to degrade xylan and convert the resulting xylose to ethanol and other products are given. 4. Bioassay to screen bacterial isolates for Arginine Deiminase a potent inhibitor of cancer cell growth, and potential inducer of autoimmunity diseases Introduction Very little is known about Arginine Deiminase (AD), an enzyme which catalyzes the irreversible hydrolysis of arginine to citrulline and ammonia. The enzyme has been found in a number of organisms such as bacteria, Mycoplasma spp., unicellular green algae, and yeasts, as well as the protozoan Tetrahymena pyriformis, and higher eukaryotes. Energy requirements for Bacteria are heterogeneous. Certain strains can utilize carbohydrates, whereas others, containing bacteria of human origin, do not. Arginine Deiminase is the first enzyme in the arginine degradation pathway to ornithine via citrulline, which yealds in a gain of ATP as energy source. Using this perhaps simplest energy-generating pathway known, most if not all of the ATP regeneration in rapidly growing cultures of Salmonella faecalis and Mycoplasma arthritidis is made. Some bacterial strains are exceedingly rich in AD, which can be up to 10% of soluble cell protein. In immune-competent or deficient organisms infections with bacteria can produce various diseases including Crohn`s disease (chronic intestinal infection), Helicobacter pylory ulcus, arthritis, pneumonia and sepsis. The mechanisms for pathogenesis are not well understood, but they could be the result of a close interaction of the microorganism with the host cell. For the Arginine Deiminase it could be shown, that it depletes arginine from local tissue and thereby inhibits proliferation of the host cell, including immune and vascular endothelial cells. The inhibition of human peripheral blood mononuclear cell proliferation is also associated with Arginine Deiminase activity. Moreover, it could be shown that Arginine Deiminase from Mycoplasma argini inhibits proliferation of various cultured cells by arresting the cell cycle in G1 and/or S phase. Higher AD concentrations lead to subsequent apoptosis. Interestingly in some cases it could be shown that the growth arrest was enhanced in arginine free media. Thus, special forms of AD could directly induce cell death by a not know mechanism of interaction with the cancer cell. Probably the best known and clinically most important growth-inhibitory (cytostatic) enzyme is Asparaginase. It is used successfully for treatment of acute lymphatic leukemia and certain solid malignancies. However, Asparaginase treatment is accompanied by serious side effects including anaphylactic shock, coagulopathies as well as liver and pancreatic toxicity. These properties outline the need for alternative treatments. Arginine Deiminase has been reported to have just a few side effects in animal studies meriting its further clinical evaluation. AD may also play an important role in pathogenesis of autoimmune diseases. Potent down regulation of regulatory T cells may allow bacterial resistance, thus potentiating other proinflammatory sequelae and increasing the potential for autoimmunity. Moreover it could be shown that in 80% of Rheumatoid Arthritis (RA), patients have antibodies against citrullinated peptides, argueing for Arginine Deiminase activity in these patients. Screening for Arginine Deiminases Searching for bacterial strains, which are high in AD activity is the aim of the demonstration. Bacterial AD will be detected by measuring their ability to hydrolyse radioactive arginine to citrulline. 1. Sampling The bacteria will be sampled from primate and rodent saliva, faeces, skin infections, gingiva and palata platina. 2. Selection criteria and axenic cultures Samples from different species and sampling locations will be plated on Blood Agar, for detection of their hemolytic potential, which correlates in most cases with pathogenic potential. (Other selection criteria: Arginine free media…). Axenic cultures of isolated strains will be obtained by series of subcultivations. Cell extracts of pure strains will be tested for AD enzyme activity. 3. Enzyme Assay The assay is based on the radio-autographic detection of H3-Citrulline. Bacterial cell extracts containing AD convert H3-Arginine to H3-Citrulline. After incubation the reaction product can be enriched by soil chromatography (Dowex X-8) where arginine is binding to the matrix, while radioactive citrulline is in the flow through. The eluate can be spotted on Whatmann paper, covered by x-ray film and developed. Positive spots can be correlated to the original sample. Physiological and molecular genetic characterization and identification of the isolates. Deposition of the isolated bacterial strains in a culture collection. Immunological detection The bacterial strain corresponding to the positive screening signal will be analyzed by Immunoblotting using primary Anti AD Antibodies IgY from chicken (provided by the scientist). Immune precipitation Is the positive strain confirmed, the protein (AD) can be precipitated using antibodies. Future Investigations Pure protein obtained from selected bacterial strains can be tested, if they are targets of autoimmune response in RA patients sera by Immunoblotting. Bacterial species producing targeted AD are of mayor interest for further investigations, because they are possible inducers for autoimmunity. 5. Xenobiotics: Enrichment and Isolation of Herbicide degrading Bacteria Biological and geochemical processes produce enormous quantities of organic compounds with a great diversity of structures. Nearly every one of these compounds can be utilized by some microorganisms as a source of energy. Even novel artificial compounds, produced by chemical synthesis (xenobiotics), for industrial or agricultural purposes can be used by some microorganisms to generate energy and/or cell building blocks. The number of xenobiotics released into the environment on a large scale, is still growing. Some, as compounds of fertilizers, pesticides and herbicides are even distributed by direct applications and many of the toxic xenobiotics are progessively concentrated in each link of a food chain (biomagnification) such as the fat soluble PCBs. Several bacterial strains (mainly Pseudomonads) isolated from soil, carry catabolic plasmids, that specify degradative pathways to degrade xenobiotics. The majority of these plasmids are self-transmissable and represent a pool of metabolic potential, available to many strains in a microbial community, through interspecies transfer of genetic information. 6. Wastewater Treatment: Enrichment and Isolation of Heavy Metal Resistant and/or Heavy Metal Accumulating Bacteria Microorganisms imobilize metal ions by either active or passive processes. For example bacteria, that use sulfate as a terminal electron acceptor, actively produce and excrete ion-sulfides that form insoluble complexes that precipitate in solutions. In contrast biosorption, where metal ions strongly bind to bacterial cell walls and to polymeric substances, secreted by bacterial cells is a passive process, seen with both living and dead cells. The binding properties of such biosorbents derive from negatively charged functional groups (carboxylate, phosphate) on the cell walls and exopolymers of the microorganism. Biosorbents effectively remove low concentrations of heavy metals (such as Cu2+, Zn2+, Cd2+, Ni2+ and Pb2+) in the presence of high concentrations of alkaline earth metals (Ca2+ and Mg2+). The picture below shows a thin section of the bacterium Alcaligenes faecalis surrounded by a thick slime capsule of excreted polyglucose coated with heavy metal ions. Objectives Description Deliverables Partner Biodiversity