Microbial Metabolism and Applications_Virulence factors-L20-24_OCT 14-22_2024 PDF

L20-24: Microbial Metabolism & Applications Dr. Ruchi Jain Dey Department of Biological Science BITS Pilani, Hyderabad Oct 14th-16th and 22nd Oct, 2024, Location G101  Handout https://docs.google.com/document/d/1uNPxs5JXS3jefuh3- potkAVLXCNQxTX...

L20-24: Microbial Metabolism & Applications Dr. Ruchi Jain Dey Department of Biological Science BITS Pilani, Hyderabad Oct 14th-16th and 22nd Oct, 2024, Location G101  Handout https://docs.google.com/document/d/1uNPxs5JXS3jefuh3- potkAVLXCNQxTXX/edit?usp=sharing&ouid=111417764931967095520&rtpof=true&sd=true 3. Presentation Variabl 20% [60M] To be 20 % CB and e - Presentation announced (60M) Announced [20M] Quizzes (4) - Class participation /Attendance [10M] - 4 announced quizzes (Best 3 out of 4) [30M, CB] Presentation Topics https://docs.google.com/spreadsheets/d/1vF3uO_noFcRsKrm1Ycr- ZhuWSS60XkmFn845zO6Q-5A/edit?usp=sharing  Chalk and board discussions have also been added in form of notes and pictures  Purpose of Metabolism apart from energy and replication Microbial metabolism serves various functions beyond energy generation and replication. These processes allow microbes to adapt to different environmental conditions, interact with other organisms, and contribute to ecological cycles. Below are key contexts where microbial metabolism plays a role: 1. Environmental Adaptation Microbial metabolism enables adaptation to diverse environments, including extreme conditions like high temperature, pressure, salinity, and pH. Microbes can modify their metabolic pathways to survive under stress or nutrient limitation. Example: Thermophiles like Thermus aquaticus utilize specialized enzymes (e.g., Taq polymerase) to survive and metabolize at high temperatures in hot springs. 2. Defense Mechanisms Microbes produce metabolites that protect them from environmental threats, such as antibiotics and toxins produced by competing organisms or immune responses. Example: Streptomyces species produce antibiotics (e.g., streptomycin) through secondary metabolism to inhibit the growth of competing microbes in the soil. 3. Symbiotic Relationships Metabolism is crucial in symbiotic relationships, where microbes exchange nutrients with hosts or other microbes. This can support the growth of both parties and is often vital for their survival. Example: In the human gut, Bifidobacteria ferment dietary fibers, producing short-chain fatty acids (e.g., butyrate) that nourish colon cells and support immune regulation. 4. Biosynthesis of Cellular Components Microbial metabolism facilitates the synthesis of essential macromolecules such as proteins, lipids, nucleic acids, and carbohydrates, which are crucial for cell structure, function, and repair. Example: Amino acid biosynthesis: Some bacteria, such as Escherichia coli, synthesize all 20 amino acids from basic carbon sources, enabling their survival in environments where these nutrients are scarce. Cell wall synthesis: In Staphylococcus aureus, metabolism contributes to the production of peptidoglycan, a critical component of bacterial cell walls, aiding in cell integrity and division. 4. Bioremediation Certain microbes can metabolize pollutants and toxic compounds, in order to survive toxic environment. But in turn help to detoxify and restore contaminated environments. This application is essential in environmental cleanup. Example: Pseudomonas putida can metabolize aromatic hydrocarbons, such as toluene, found in oil spills, aiding in bioremediation efforts. 5. Biosynthesis of Complex Molecules Metabolism supports the synthesis of complex biomolecules necessary for microbial structure and function, such as vitamins, amino acids, lipids, and polysaccharides. These products can be essential for both the microbe and other organisms. Example: E. coli produces vitamin K2 (menaquinone) as part of its metabolic processes, which is important for blood clotting in humans. 6. Quorum Sensing and Communication Microbial metabolism plays a role in producing signalling molecules involved in quorum sensing, which is crucial for coordinating activities like biofilm formation, virulence, and resource sharing among microbial communities. Example: Vibrio fischeri uses quorum sensing to regulate bioluminescence when cell density reaches a threshold in symbiosis with certain marine animals. 7. Metabolite Exchange in Microbial Consortia In microbial communities, different species exchange metabolic products to sustain community functions, including nutrient cycling and resilience in complex environments. Example: In the nitrogen cycle, Nitrosomonas converts ammonia to nitrite through nitrification, which is further metabolized by Nitrobacter to produce nitrate, essential for plant uptake.  Detailed Examples of Multi-faceted metabolism Adaptation to Environmental Stresses Detoxification and Resistance to Toxins Metabolic pathways allow microbes to adapt to changing Microbial metabolism can neutralize harmful substances or environmental conditions, including nutrient availability, modify toxic compounds, which is critical for survival in temperature shifts, oxidative stress, and osmotic pressures. hostile environments or during antibiotic exposure. Example: Example: Osmoregulation in Halophiles: Halophilic archaea, such Antibiotic resistance in Mycobacterium tuberculosis: as Halobacterium, utilize compatible solutes (e.g., potassium The bacterium’s metabolism enables the production of ions) to maintain osmotic balance in high-salt environments, enzymes like β-lactamase that degrade antibiotics, conferring ensuring cellular stability and metabolic function. resistance to β-lactam antibiotics. Heat shock proteins in E. coli: During heat stress, Sulfate-reducing bacteria: These bacteria reduce toxic metabolic processes activate the production of heat shock heavy metals, such as mercury, through metabolic proteins that prevent protein misfolding and aggregation, processes, converting mercury into less harmful forms like ensuring cellular survival. methylmercury. Symbiotic Relationships and Nutrient Cycling Defense Mechanisms (Production of Antibiotics and Microbial metabolism often supports mutualistic and Toxins) symbiotic relationships with plants, animals, and other Some microbes metabolize secondary metabolites that microbes, influencing global nutrient cycles (e.g., carbon, function as antibiotics, toxins, or other defensive compounds nitrogen, sulfur). to inhibit the growth of competing microbes or defend against Example: predators. Nitrogen fixation by Rhizobium species: These bacteria Example: metabolize atmospheric nitrogen (N₂) into ammonia (NH₃) via Production of Penicillin by Penicillium fungi: This nitrogenase enzyme, benefiting plants by providing usable antibiotic is a metabolic byproduct used to inhibit the growth nitrogen for growth. of competing bacteria. Methanogenesis by Methanogens: In anaerobic Toxin production by Clostridium botulinum: The environments, archaea metabolize carbon compounds into bacterium produces botulinum toxin through specialized methane, contributing to carbon cycling in ecosystems like metabolic pathways, which is one of the most potent wetlands or the rumen of herbivores. neurotoxins known. Bioremediation and Environmental Metabolism Energy Harvesting from Extreme Environments Microbial metabolism is harnessed for bioremediation, where Some microbes thrive in extreme environments by metabolizing microbes metabolize pollutants and toxic compounds, unusual energy sources such as sulfur, methane, or metals, transforming them into less harmful substances. enabling life in harsh conditions where other organisms cannot Example: survive. Hydrocarbon degradation by Pseudomonas putida: This Example: bacterium can metabolize organic pollutants such as oil spills, Sulfur metabolism in Acidithiobacillus ferrooxidans: This breaking down hydrocarbons into carbon dioxide and water. bacterium derives energy by oxidizing sulfur compounds, playing a Plastic degradation by Ideonella sakaiensis: This role in acid mine drainage and bioleaching of metals. bacterium metabolizes polyethylene terephthalate (PET), a Methane consumption by Methanotrophs: These bacteria type of plastic, into simpler monomers, aiding in waste metabolize methane as a carbon and energy source, contributing management and plastic recycling. to greenhouse gas mitigation in soil and water ecosystems. Biofilm Formation and Pathogenicity Metabolic processes in microbes are closely linked to biofilm formation, which enhances pathogenicity and resistance to environmental pressures, including immune responses and antibiotics. Example: Biofilm formation in Staphylococcus epidermidis: The bacterium metabolizes carbohydrates and produces extracellular polymeric substances, forming biofilms that are highly resistant to antibiotics and immune attack, often found on medical devices like catheters. Dental plaque formation by Streptococcus mutans: This bacterium metabolizes sugars into lactic acid, leading to acid production that demineralizes teeth and contributes to the formation of dental plaque.  Metabolism & Biofilms Youtube Videos: https://www.youtube.com/watch?v=1YKeLMXuBoU [Beneficial Biofilm Bacteria - waste water treatment] 11 12  Why Microbes Talk? ✔ Survive by establishing a homeostasis between microbial neighbors and local environments. ✔ Response to environmental stimuli ✔ Morphological and developmental processes of the organisms themselves and their neighbors. ✔ Shapes the entire habitat of these organisms. 13 ❖ Cell-to-cell contact ❖ Contact-independent ⮚ Signals are dispersed much faster ⮚ reach many neighboring cells and communities ⮚ metabolic exchange factors (Quorum-sensing factors) -nutrients or cues to neighboring microbes ⮚ control the behavior of the larger microbial community leading to behavior as a multicellular entity. ❖ Heavy investment- 17–42% of the genes of bacteria are dedicated to microbial interactions 14 Cellular Networking : How a bacteria talks through Metabolites 15 Talk to host 16 Omics : Revolutionizing the way we look at disease and the way we treat 17 Current multi-omics technologies to understand biology 18 Key Enzymes of Microbes 19  Exoenzymes – transported extracellularly, where they break down large food molecules or harmful chemicals; cellulase, amylase, penicillinase Endoenzymes – retained intracellularly & function there 20  Exoenzymes – A virulent factor Youtube videos: https://www.youtube.com/watch?v=XBTkgu8peEY Exoenzymes – A virulent factor What is toxic shock syndrome? Youtube link: https://www.youtube.com/watch?v=hSYKmbIVpaY https://www.youtube.com/watch?v=WEjqRCloqdk  Bacterial Toxins 1. Nucleases 2. Cell-envelope degrading toxins 3. Pore-forming toxins 4. Cell-function inhibitors- destroys ATP, destroy cofactors https://sarahs-world.blog/bacteria-deliver-toxins/ Toxin s  YouTube link: https://www.youtube.com/wat ch?v=gnZEge78_78 Most dangerous: Clostridium tetani (Tetanus), Corynebacterium diphtheriae (Diphtheria). 1 mg of intravenous endotoxins can have lethal consequences. Endotoxin can be inactivated when exposed at temperature of 250º C for more than 30 minutes or 180º C for more than 3 hours (28, 30). Acids or alkalis of at least 0.1 M strength can also be used to destroy endotoxin in laboratory scale Coagulase Test The coagulase test is one way to differentiate the highly pathogenic S. aureus from the other less pathogenic staphylococcal species on the human body. S. aureus is a coagulase-positive organism whereas all the other staphylococcal species that colonize humans are coagulase negative.  Practice question: Identification of bacteria based on Starch test: Which one makes amylase? A. Staphylococcus epidermis B. Bacillus subtilis  Lipase test ✔Lipase is an exoenzyme that hydrolyzes lipids into fatty acids and glycerol. In this photograph, two organisms were streaked on a plate of spirit blue agar. ✔This agar contains lipids and spirit blue dye. If a microorganism produces lipase, the fats around the streak are decomposed, causing a clear zone to appear. ✔The spirit blue dye also migrates through the plate toward the region lacking complete lipids, resulting in the appearance of a dark blue halo around the organism. Does S. epidermidis produces lipase or E. coli ?. Lecithinase or Nagler’s test Protease test Proteases- hydrolyze proteins into polypeptides and amino acid subunits. Two examples of digestive proteases: A.Caseinase Caseinase is an exoenzyme that hydrolyzes the milk protein casein. If in a plate of skim milk agar bacteria is streaked, as the casein is hydrolyzed around the streak, a clear halo appears. Which one is Gelatinase Positive P. vulgaris A or B ?  Constitutive enzymes – always present, always produced in equal amounts or at equal rates, regardless of amount of substrate; enzymes involved in glucose metabolism Induced enzymes – not constantly present, produced only when substrate is present, prevents cell from wasting resources 40  Synthesis or condensation reactions – anabolic reactions to form covalent bonds between smaller substrate molecules, require ATP, release one molecule of water for each bond Hydrolysis reactions– catabolic reactions that break down substrates into small molecules, requires the input of water 41 42 Metabolic pathways 43  Industrial Application of Microbial Metabolism (For basic metabolic pathway refer to Biochemistry chapters) Metabolic strategies Pathways Final e- involved acceptor ATP yield Aerobic Glycolysis, O2 38 respiration TCA, ET Anaerobic Glycolysis, NO3-, So4-2, variable respiration TCA, ET CO3-3 Fermentation Glycolysis Organic 2 molecules 45  Fermentation Any metabolic process that releases energy by Incomplete oxidation of glucose or other carbohydrates in the absence of oxygen Does not require ETC (electron transport chain) Uses organic compounds as terminal electron acceptors Yields a small amount of ATP compared to glycolysis Examples – ✔Production of ethyl alcohol by yeasts acting on glucose ✔Formation of acid, gas & other products by the action of various bacteria on pyruvic acid 47 Fermentation 48 Fermentation: An important metabolic pathway How to identify Fermentative and Non-fermentative microbes? Waste product of one microbe can be used by carbon and energy source by another microbe Vinegar Holes in swiss cheese  Types of Metabolism (Self study- Basics about different carbon source and different types of metabolism found in microbes. Symbiotic Microbial associations Symbiotic – organisms live in close nutritional relationships; required by one or both members Mutualism – obligatory, dependent; both members benefit Commensalism – commensal member benefits, other member not harmed Parasitism – parasite is dependent and benefits; host is harmed 55 Microbial associations Non-symbiotic – organisms are free-living; relationships not required for survival Synergism – members cooperate and share nutrients Antagonism – some member are inhibited or destroyed by others 56 The digestive tracts of a herbivore (koala) The koala′s intestines are much longer, an adaptation that enhances processing of fibrous, protein–poor eucalyptus leaves from which it obtains virtually all its food and water. The koala′s caecum—at 2 m, the longest of any animal of equivalent size— functions as a fermentation chamber where symbiotic bacteria convert the shredded leaves into a more nutritious diet. 67 Chemical composition of cytoplasm 70% water 30% chemicals ✔ Proteins ✔ Small molecules ✔ RNA ✔ Phospholipids ✔ DNA ✔ Polysaccharides 96% of cell is composed of 6 elements ⮚ Carbon ⮚ Hydrogen ⮚ Oxygen ⮚ Phosphorous ⮚ Sulfur 72 73 Carbon 75 Nitrogen Main reservoir is nitrogen gas (N2) 79% of earth’s atmosphere is N2 Nitrogen is part of the structure of proteins, DNA, RNA & ATP – these are the primary source of N for heterotrophs Some bacteria & algae use inorganic N nutrients (NO3-, NO2-, or NH3) Some bacteria can fix N2 Regardless of how N enters the cell, it must be converted to NH3, the only form that can be combined with carbon to synthesis amino acids, etc. 76 Oxygen major component of carbohydrates, lipids and proteins plays an important role in structural & enzymatic functions of cell component of inorganic salts (sulfates, phosphates, nitrates) & water O2 makes up 20% of atmosphere essential to metabolism of many organisms 78 Hydrogen major element in all organic compounds & several inorganic ones (water, salts & gases) gases are produced & used by microbes roles of hydrogen maintaining pH forming H bonds between molecules serving as the source of free energy in oxidation- reduction reactions of respiration 79 Phosphorous main inorganic source is phosphate (PO4-3) derived from phosphoric acid (H3PO4) found in rocks & oceanic mineral deposits key component of nucleic acids, essential to genetics serves in energy transfers (ATP) 80 Sulfur widely distributed in environment, rocks, sediments contain sulfate, sulfides, hydrogen sulfide gas and sulfur essential component of some vitamins and the amino acids: methionine & cysteine contributes to stability of proteins by forming disulfide bonds 81 Important mineral ions Potassium Sodium Calcium Magnesium Iron 82 Growth factors organic compounds that cannot be synthesized by an organism & must be provided as a nutrient essential amino acids, vitamins 83  Carbon Energy source source photoautotrophs CO2 sunlight chemoautotrophs CO2 Simple inorganic chemicals photoheterotrophs organic sunlight chemoheterotrophs organic Metabolizing organic cpds 84 Transport mechanisms Passive transport –do not require energy, substances exist in a gradient and move from areas of higher concentration towards areas of lower concentration Diffusion Osmosis - water Facilitated diffusion – requires a carrier Active transport – require energy and carrier proteins, gradient independent Carrier-mediated active transport Group translocation – transported molecule chemically altered Bulk transport – endocytosis, exocytosis, pinocytosis 85 diffusion 86 osmosis 87 88 passive transport 89 Active transport 90 Bulk transport 91