Microbiology Lecture Reviewer PDF

Introduction: Microorganisms or Microbe ​ Usually smaller than 1mm in diameter ​ Often unicellular ​ Lack differentiated tissues Two Major Classes of Cells ​ Prokaryotic- Has nucleoids ​ Eukaryotic- Has nucleus and membrane-bound organelles in cytoplasm Origins of Microbiology...

Introduction: Microorganisms or Microbe ​ Usually smaller than 1mm in diameter ​ Often unicellular ​ Lack differentiated tissues Two Major Classes of Cells ​ Prokaryotic- Has nucleoids ​ Eukaryotic- Has nucleus and membrane-bound organelles in cytoplasm Origins of Microbiology ​ Robert Hooke (1665) - Cells ​ Antonie van Leeuwenhoek (1674) - Animalcules - Father of Microbiology ​ Louis Pasteur (19th Century) a.​ Fermentation - Sour vats-rod-shaoed microorganisms > lactic acid - Alcohol > yeast b.​ Spontaneous Generation - Life arises from non-living matter - Pasteur was an opponent of this theory Joseph Lister- Aseptic Technique - Pasteurization c.​ Other Discoveries - Vaccines for: ​ Anthrax ​ Cholera ​ Rabies d.​ Hand washing was advocated as prevention of infection, but there was no basis Process of Pasteurization 1.​ Non Sterile liquid poured in a flask 2.​ Neck of flask drawn out in flame 3.​ Liquid sterilized by extensive heating (Steam forced out open end) 4.​ Liquid cooled slowly > In a few days flask neck is contaminated (Dust and microorganisms from air trapped in bend) 5.​ Liquid remains sterile indefinitely/Flask is tipped gently > Microbe-laden dust mixes with sterile liquid > Liquid putrefies ​ Robert Koch (19th Century) a.​ Koch's Postulates b.​ Koch and tuberculosis - Causative = M. tuberculosis - Also discovered cholera is caused by V. cholerae - Colonies have distinct shape, size, color Koch's Postulates 1.​ The suspected pathogen must be present in all cases of the disease and absent from healthy animals ​ Lab tools- Microscopy and Staining 2.​ The suspected pathogen must be grown in pure culture ​ Lab tools- Laboratory cultures 3.​ Cells from a pure culture of the suspected pathogen must cause disease in a healthy animal ​ Lab tools- Experimental animals 4.​ Suspected pathogen must me reisolated and shown to be the same as the original ​ Lab tools- Laboratory reisolation and culture ​ Martinus Beijerinck (20th century) a.​ Enrichment culture technique b.​ Rhizobia in root culture of legumes c.​ Sulfate-reducing bacteria d.​ Sulfur-oxidizing bacteria e.​ LAB f.​ Green algae g.​ Various anaerobic bacteria h.​ Tobacco mosaic virus ​ Sergei Winogradsky (20th century) a.​ Beggiatoa is a chemolithotroph b.​ Chemo- Chemical compounds as energy source c.​ Lithotroph- Rock eaters; inorganic electron source d.​ C. pasteurianum- Anaerobic N-fixing bacteria e.​ Azotobacter- Aerobic N-fixing bacteria ​ Carl Worse (20th century) a.​ Tree of life b.​ Ribosomal RNA (rRNA) i.​ Universally distributed ii.​ Functionally constant iii.​ Highly conserved iv.​ Adequate length Importance of Microbiology ​ Agents of disease ​ Agriculture and human nutrition ○​ Rhisobia: N2 > NH3 ○​ Gut microbe: Digestion ​ Food ○​ Fermentation ​ Industry ○​ Biofilm ○​ Antibiotics, enzyme, and certain chemicals ○​ Bioremediation- Wastewater treatment Microbial Diversity: Early Earth and Life Forms ​ Earth formed about 4.5 BYA Eons: 1.​ Hadean (Anoxic Atmosphere) ○​ Earth Sterilization: ​ Formation of earth ​ Formation of crust and oceans ○​ Origins of cellular life 2.​ Archaean (Anoxic Atmosphere) ○​ Divergence of Bacteria and Archaea ○​ Origins of anoxygenic photosynthesis ○​ Origins of cyanobacteria and oxygenic photosynthesis 3.​ Proterozoic ○​ Start of Great Oxidation Event ○​ Ozone Shield Forming ○​ Evidence for multicellular eukaryotes ○​ Start of Cambrian Explosion 4.​ Phanerozoic ○​ Origins of first animals ○​ Extinction of dinosaurs ​ Early Earth was a sterile planet ​ Liquid water is a requirement for life ○​ Volcanic outgassing ○​ Icy comets and asteroids Evidences: 1.​ Stratospheric Observatory for Infrared Astronomy (SOFIA) a.​ Observed the comet Wirtanen in December 2018 2.​ Jack Hills Zircon a.​ 4.4 BY b.​ Oldest known mineral 3.​ Oldest surviving sedimentary rock a.​ Southern Greenland b.​ 3.86 BY Early Life Forms: ​ Anaerobes ​ Thermophilic H2 Utilizing ○​ 2H2 + CO2 > CO2 + (CH2OH)- carbohydrates ​ Led to chemoorganotrophs ○​ Methanogens ​ Hyperthermophiles ○​ 80-113 C ○​ 0-9.0 pH ○​ Thermoproteus ○​ Methanopyrus ○​ Pyrodictium and Pyrolobus ​ LUCA ○​ Chemolithotrophs ○​ Anaerobic ○​ Lived in high temperature ○​ Rich in S and Fe Great Oxidation Event ​ 2.6 BYA ​ Anoxygenic photosynthesis by purple and green bacteria ○​ 6CO2 (Carbon Dioxide) + 12H2O (Water) + Light Energy > C6H12O6 (Glucose) + 6O2 (Oxygen) + 6H2O (Water) ​ Oxygenic photosynthesis by cyanobacteria  ○​ Redox ○​ O2 ○​ O3 ○​ CO2 (Carbon Dioxide) + 2H2A (Electron Donor) + Light Energy > [CH2O] (Carbohydrate) + 2A + H2O (Water) Evidences: ​ Stromatolites ○​ Layered rocks Diversity of Domain Bacteria ​ Phototrophs Phyla: 1.​ Firmicutes 2.​ Proteobacteria 3.​ Chloroflexi 4.​ Cyanobacteria 5.​ Chlorobi 6.​ Acidobacteria ​ Has photochemical center Oxygenic: ​ Type 1 RC (FeS) ​ Chlorobi, Firmicutes, and Acidobacteria ​ Type 2 RC (Quionones) ​ Chloroflexi and Proteobacteria Anoxygenic: ​ HS2 ​ Proteobacteria and Chlorobi ​ Organic ​ Proteobacteria and Chloroflexi Phylum Cyanobacteria ​ Gram-negative (thin walls) ​ Type 1 and 2 RC ​ Calvin-Benson-Basham Cycle ​ Phycobilisomes Phylum Chlorobi ​ Green sulfur bacteria ​ Obligate anaerobic phototrophs ​ Type 1 RC and Chlorosomes coupled to Fenna-Matthews-Olson proteins ​ Bchl c (745-755 nm), d (705-740 nm), and e (716-726 nm) Phylum Proteobacteria ​ Purple bacteria ​ Gram-positive ​ Carotenoids ​ Bchl a (805, 830-890 nm), b (835-850 nm, 1020-1040 nm) ​ Can be sulfur and non-sulfur Phylum Chloroflexi ​ Filamentous ​ Anoxygenic ​ Can switch metabolism ​ Chloroflexales ​ Type 2 RC Phylum Firmicutes ​ Obligate anaerobes ​ Photoheterotrophic or Chemoheterotrophic ​ Endospores Phylum Acidobacteria ​ Chloracidobacterium thermophilum Bacterial Cell Wall ​ Peptidoglycan structural sugars: ○​ N-acetylglucosamine (NAG) ○​ N-acetylmuramic acid (NAM) ​ Two types of bacteria based on cell wall composition: ○​ Gram-positive ​ Lipoteichoic acid and teichoic acid ​ Thick peptidoglycan ​ Small periplasmic space ​ Secretes exoenzymes ○​ Gram-negative ​ Outer membrane ​ Thin peptidoglycan ​ Thick periplasmic space Significance: ​ B-lactam ring Diversity in Shape and Arrangement Diversity in Flagellar Arrangement Diversity in Domain Archaea ​ Can live in extreme and normal conditions ​ Extremophiles- Microorganisms that inhabiit extreme conditions  ○​ Extremophilic Organisms- One or more extreme conditions ○​ Extremotolerant Organisms- tolerate extreme conditions Hyperthermophiles ​ Often found in solfataric fields (Volcanoes) ​ Pyrodictium occultum (110C) Ferroplasma ​ Cell wall-less ​ Iron-oxidizing archaeon in an acidic environment ​ Caldarchaetidylyceroo tetraether ​ F. acidiphilum Methanogens ​ Phylum Euryarchaeota ​ Live in normal conditions ​ Produces biological methane via anaerobic conditions ​ Might have contributed to the largest extinction in history Halophilic Archaea ​ In hypersaline environments ​ Pigments due to carotenoids Diversity of Domain Eukarya ​ Protist ○​ Diplomonads ​ Flagellated, unicellular organisms ​ Two nuclei ​ Mitosomes ​ Giardia ○​ Parabasalids ​ Parabasal body ​ T. vaginalis ​ Can be obligate symbiont or parasite of vertebrates ○​ Euglenozoans ​ Flagellated ​ Free-living, symbiotic, or parasitic ​ Euglena ○​ Alveolates ​ Has alveoli ​ Ciliate = Cilia ​ Dinoflagellates = Flagella ​ Apicomplexans = Has apicoplast ​ Fungal- Decomposers and recyclers ○​ Zygomycetous ​ Has coenocytic hyphae ​ Sexual reproduction produces zygospores  ○​ Glomeromycotina ​ Arbuscular mycorrhizal fungi ○​ Basidiomycota ​ Also called club fungi ​ Has basidium that produces basidiospores ○​ Ascomycota ​ Sac fungi ​ Has ascus ​ Sac-shaped reproductive structure ​ Can have yeast or mold morphology Archaeplastida ​ Large group ​ Red Algae, Green Algae, and land plants ​ Red and Green Algae = primary endosymbiotic events ​ Other protists containing chloroplasts = secondary endosymbiosis ​ Red and Green Algae- Contain chlorophylls and carry ou oxygenic photosynthesis Red Algae Polysiphonia, Cyanidium, Galdiera ​ Also called rhodophytes ​ Mainly inhabit marine environment ​ Few are found in freshwater and terrestrial habitats ​ Unicellular and multicellular ​ Some are macroscopic Major Properties: 1.​ Phototrophic 2.​ Chlorophyll a 3.​ Chloroplasts lack chlorophyll b, but contain phycobiliproteins (Major light-harvesting pigment of cyanobacteria) 4.​ Reddish color comes from phycoerythrin (Accessory pigment that masks the green color of chlorophyll) 5.​ Phycocyanin and allophycocyanin is also resent in phycobilisomes (light-harvesting component/antenna of Cyanobacteria) 6.​ Greater depths in aquatic habitats > Less light > More phycoerythrin > Darker Red 7.​ Shallow-dwelling species > Less phycoerythrin > Green 8.​ Inhabit marine environments 9.​ Multicellular 10.​Lack flagella  11.​Some are seaweeds 12.​Source of agar and carrageenans (Thickening and stabilizing agents used in the food industry) Genus Porphyra- Are harvested, dried, and used in wraps in sushi Different species in Red Algae are: ​ Filamentous ​ Leafy ​ Coralline or coral-like (If they deposit calcium carbonate) ○​ Coralline Red Algae- Play an important role in the development of coral reefs, as well as strengthening them against wave damage Genus Polysiphonia ​ Filamentous ​ Multicellular ​ Branched ​ Found worldwide in marine environment (Near shore attached to rocks, other algae, and man-made surfaces) ​ Nearly 200 species are recognized ​ Undergoes a complex life cycle (Alternation generation) Cyanidium, Galdiera, and Relatives ​ Cyanidiales ○​ Genera Cyanidium, Cyanidioschyzon, an Galdiera ○​ Unicellular ○​ Live in hot, acidic, metal-rich hot springs ○​ 30-60°C ○​ 0.5-4.0 pH ○​ No other phototrophic microorganisms, including anoxygenic phototrophs can exist under these extreme conditions Cyanidioschyzon merolae ​ Unusually small for eukaryotes ​ 1-2 μm ​ Approximately 16.5 Mbp is one of the smallest genome known for a phototrophic eukaryote Galdieria ​ Contains at least 75 genes from horizontal transfer from various prokaryotic sources ​ 25 μm ​ More blue than red due to its phycobilin containing mainly phycocyanin, rather than phycoerythrin ​ Some key genes acquired:  ○​ Protection from salt stress ○​ Metal toxicity ○​ Toughened cytoplasmic membrane against heat and acidity of its habitat ○​ Grow in darkness Green Algae ​ Chlamydomonas and Volvox ​ Also called chlorophytes ​ Chlorophyll a and b ​ Lack phycobiliproteins and do not develop red or blue-green colors ​ Photosynthetic elements are similar to plants and close to them phylogenetically ​ Two groups: ○​ Chlorophytes (Chlamydomonas and Dunaliella) ​ Microscopic ○​ b. Chlorophyceans (Chara) ​ Macroscopic ​ Most inhabit freshwater others in moist soil or in snow (Pink color) ​ Other live with fungi in a symbiotic relationship (lichens) ​ Unicellular ​ Filamentous (Individual cells arranged end to end) ​ Colonial (Aggregates of cells) ​ Multicellular (Ulva) ​ Complex life cycle (Asexual and sexual reproductive stages) Very Small Green Algae and Colonial Green Algae ​ Smallest eukaryotes known is Ostreococcus tauri ○​ Common unicellular species of marine phytoplankton ○​ 2 μm in diameter ○​ Contains the smallest genome of any phototrophic eukaryote (Approx. 12.6 Mbp) ○​ Model organism for research on evolution of genome and specialization in eukaryotes ​ Colonial level is Volvox ○​ Forms colonies composed of several hundred flagellated cells ○​ Some are motile and primarily carry out photosynthesis ○​ Others specializes in reproduction ○​ Cells in a colony are interconnected by a thin strand of cytoplasm, helping them swim in a coordinated fashion ○​ Long term model research on genetic mechanism controlling multicellularity and distribution of functions among cells in multicellular organisms ​ Some colonial green algae have potential as sources of biofuels (Botryococcus braunii) ○​ Excretes long chain of hydrocarbons that have the consistency of crude oil (C30-C36) ○​ About 30% of its cell dry weight consists of this petroleum  ○​ There's been heightened interest in using this and other oil producing algae as renewable sources of petroleum ○​ Biomarker studies have shown that some known petroleum reserves originated from green algae like B. braunii Endolithic Phototrophs ​ Some green algae grow inside rocks ​ Inhabit porous rocks (Those containing quartz) ​ Typically found in layers near rock surface ​ Most common in dry environments (Deserts or cold, dry environments like Antarctica) ​ In McMurdo Dry Valleys of Antarctica where temp, and humidity are low, living within a rock has its advantages ​ Rocks are heated by the sun ​ Water from snowmelt can be absorbed and retained for relatively long periods providing moisture ​ Water absorbed by porous rocks make them more transparent (Funneling more light) ​ Cyanobacteria and various green algae ​ Free living phototrophs ​ Can coexist with fungi in lichen communities ​ Metabolism and growth within rocks, weathers the rock allowing water to enter, freeze, and thaw ​ Decomposing rocks produces crude oil that can support plant and animal communities where conditions permit Microbial Growth Requirements Cell Growth ​ Binary fission ​ Budding- Unequal growth ​ Spore-formation (Streptomyces, Fungi) Process of Metabolism: a.​ Young cell at early phase of cycle  b.​ Parent cell prepares for division by enlarging the cell wall, plasma membrane, and overall volume. DNA replication then starts c.​ Septum grows inward as chromosomes move at opposite ends of the cell. Other cytoplasmic components are distributed to the two developing cells d.​ Septum is synthesized completely through cell center, creating two separate chambers e.​ Daughter cells are divided (Some species separate completely; others remain attached forming chains, doublets, or other cellular arrangements Growth Cycle ​ Refers to a time where one cell separates to form two cells ​ One generation: ○​ Cell Elongation ○​ Septum formation ○​ Completion of septum > Formation of walls > Cell separation ​ Growth Curve ​ Lag Phase- Microorganisms exhibit slow growth as it adjust to environment  ​ Exponential Phase- Growing cell population doubles at regular intervals ​ Stationary Phase- No net increase or decrease in cell number (Cryptic Growth) ​ Death Phase- Also occurs as an exponential function, but much slower than exponential growth ​ Long-Term Stationary Phase- Can last months to years Quantitative Aspects ​ Formula: 2^n (Doesn't consider initial bacterial population) ​ N= Final cell number ​ N0= Initial cell number ​ n= Number of generation ​ n= Number of generation ​ N= Final cell number ​ N0= Initial cell number  ​ g= Generation time ​ t= Durational growth ​ n= Number of generations ​ v= Division rate ​ g= Generation time ​ k= Specific growth rate ​ v= Division rate ​ g= Generation time Nutritional Requirements ​ C ○​ Chemoheterotrophs ○​ Chemoautotrophs ​ O ○​ Obligate aerobes ○​ Facultative Aerobes ○​ Obligate Anaerobes ○​ Aerotolerant Anaerobes ○​ Microaerophiles ​ N ​ S ​ P ​ Ca ​ Trace Elements (Fe, Cu, Zb, Zn) Culturing Microbes Culture Media- Nutrient solutions tailored to particular organisms to be grown  Classifications: ​ Preparation ○​ Skewed Media ○​ Plate Media ​ Chemical Composition ○​ Chemically Defined- Exact amount of organic and inorganic chemicals for a certain bacterial growth ○​ Complex Media- Exact chemical composition is unknown ​ Consistency ○​ Agar Media- Complex polysaccharide from algae (Liquefies at 100C; Solidifies at 40C) ○​ Liquid Medium- Absence of agar ​ Function ○​ Environmental Function ​ Fastidious vs Non-Fastidious Microorganism ​ Simple Media/Basal Media- Contains enough nutrients to sustain non-fastidious microorganisms (TSA, TSB) ​ Enriched Media- Has additional nutrients for fastidious microorganisms (Blood Agar) ​ Enrichment Media- Relatively increases the growth of specific organisms in the culture (Alkaline Peptone Water) ​ Selective Media- Inhibit the growth of some microorganisms, while allowing growth of others (MSA) ​ Differential Media- Has an indicator, revealing a color change whether a particular metabolic reaction occurred during growth (MacConkey Agar Plate) Aerobic Metabolism ​ Uses oxygen to make energy from food we eat like glucose ​ Stored in the form of ATP (Required for contraction of muscles or other chemical operations) ​ Occurs in mitochondria Mitochondria- Powerhouse of cell ​ If oxygen is present aerobic metabolism occurs ​ Produces a lot of ATP with small amounts of glucose ​ Glycolysis: Glucose, Glycogen, Fats, Glycerol ​ Pyruvate > Acetyl Coenzyme A ​ Krebs Cycle ​ Electron Transport Chain Glycolysis ​ Glucose is broken down into two molecules of pyruvate ​ Occurs in cytosol ​ Generates small amount of ATP and reducing elements (NADH) ​ Doesn't need oxygen ​ Can occur in aerobic and anaerobic conditions Process of Glycolysis: 1.​ Phosphate from hydrolysis of ATP reacts with the glucose, forming glucose-6-phosphate 2.​ Molecule rearranges to isomer fructose-6-phosphate 3.​ Another phosphate from hydrolysis of another ATP is added forming fructose-1, 6-bisphosphate 4.​ Fructose-1, 6-bisphosphate splits forming glyceraldehyde-3-phosphate and dihydroxyacetone phosphate (Isomers) 5.​ Dihydroxyacetone phosphate is converted by triose-phosphate isomerase into glyceraldehyde-3-phosphate 6.​ Two reactions occur: a.​ Enzyme glyceraldehyde-3-phosphate transfers a hydrogen to nicotinamide adenine dinucleotide > NADH + H+ b.​ Glyceraldehyde-3-phosphate dehydrogenase donates a phosphate to the oxidized glyceraldehyde phosphate > 1,3-diphosphoglycerate 7.​ 1, 3-diphosphoglycerate transfers phosphate to ADP through phosphoglycerokinase > ATP 8.​ Both phosphoglycerate transfers their phosphate from second carbon to third carbon > 2-phosphoglycerate 9.​ A water molecule is removed by an enzyme (enolase) from 2-phosphoglycerate > phosphoenolpyruvate 10.​Phosphate is transferred to ADP > Pyruvate and ATP Link Reaction ​ Connects glycolysis with Krebs cycle  ​ Occurs in mitochondria ​ Pyruvate is oxidized (Removes a molecule of carbon dioxide) > 2-carbon acetyl group ​ This binds with coenzyme a > Acetyl Coenzyme A ​ One of the carbons is released as CO2 Process of Link Reaction: 1.​ Pyruvate enters mitochondria 2.​ A carbon is removed, released as CO2 leaving behind the two-carbon molecule 3.​ Two-carbon molecule is oxidized, then the electron lost during this process is picked up by NAD+ > NADH 4.​ The oxidized two-carbon molecule (acetyl group) attaches to coenzyme a > Acetyl Coenzyme A Citric Acid Cycle ​ Also known as Krebs Cycle or tricarboxylic acid cycle ​ Occurs in mitochondrial matrix and uses Acetyl CoA (Biggest source of ATP) ​ Central part of cellular respiration ​ It should yield carbon dioxide, NADH, FADH2, and GTP or ATP Process of Krebs Cycle 1.​ Acetyl-CoA is bonded with oxaloacetate, 4-carbon acceptor > 6-carbon molecule (citrate)  2.​ Removal of water molecule and addition of water molecule to convert citrate to isocitrate 3.​ Oxidation of isocitrate > A-ketoglutarate (Catalyzed by isocitrate dehydrogenase) 4.​ A-ketoglutarate is oxidized > Reduction of carbon dioxide and turning NAD+ to NADH > Unstable succinyl CoA 5.​ CoA of succinyl CoA is replaced with a phosphate group in order to transfer ADP to make ATP 6.​ Oxidation of succinate > Fumarate. FAD and Two hydrogen atoms > FADH2 > Electron Transport Chain 7.​ Water molecule is added with fumarate > Malate 8.​ Regeneration of oxaloacetate through oxidizing Malate. NAD+ > NADH (Reduction) Electron Transport Chain ​ Series of protein complexes and electron carriers in inner mitochondrial membrane ​ Transfers electrons from NADH and FADH2 to oxygen > Proton gradient that drives ATP synthesis (ATP synthase) ​ Reactions: Redox, Proton pumping for gradient, and ATP synthesis via chemiosmosis ​ Reactants: NADH and FADH2, O2, ADP, and organic phosphate ​ Products: H2O from reduced oxygen, ATP, NAD+, and FAD Process of Electron Transport Chain: 1.​ NADH and FADH2 donate electrons > NAD+ and FAD+  2.​ Electron transfers to ubiquinone (Electron carrier) 3.​ Complex III pumps protons into the intermembrane space 4.​ Electrons are transferred from Complex III to Cytochrome C, and transported to Complex IV 5.​ Proton gradient drives ATP synthase 6.​ ADP and Pi > ATP Products of Aerobic Metabolism: ​ Carbon Dioxide ​ Water ​ Energy (ATP) Total Yield of Aerobic Metabolism: ​ 38 ATP (2 from Glycolysis; 36 from Electron Transport Chain) Application of Aerobic Metabolism ​ Food Production ○​ Fermentation ​ Bioremediation ○​ Toxic waste depredation ​ Wastewater treatment ○​ Biological oxygen demand reduction ​ Industrial ○​ Biofuels Anaerobic Respiration ​ Does not rely on oxygen as final electron acceptor in Electron Transport Chain ​ Other inorganic or organic molecules are used: ○​ Nitrate (NO3-) ○​ Sulfate (SO4²-) ○​ Carbon Dioxide (CO2) ​ Less efficient than aerobic respiration, but crucial for survival in oxygen-deprived environments Anaerobes ​ Includes certain bacteria, archaea, and some eukaryotic microbes that doesn't require oxygen ​ Origins traces back to Early Earth (Probiotic era- Primarily anaerobic)  Aerobic Anaerobic Mechanism Energy production in the presence Energy production in the of oxygen absence of oxygen Energy Source Organic energy and electrons Organic energy and electrons End Products CO2, H2O, ATP CO2, Reduced inorganic molecule, ATP Terminal Electron O2 (Oxygen) SO4²-, NO3-, CO2, Acceptor (ETC) Fumarate, etc. (Inorganic Molecules) ATP Yield High (30-38 ATP molecules per Low (2 ATP molecules per glucose molecule) glucose molecule) Facultative Anaerobes ​ Capable of switching aerobic, anaerobic, and fermentation depending on oxygen availability ​ This flexibility allows them to thrive in oxygen-rich and oxygen-poor environment ​ Able to tolerate oxygen exposure due to Superoxide Dismutase ○​ Neutralizes harmful Superoxide radicals, converting them to less toxic molecules (Hydrogen Peroxide) ​ This protective mechanism ensures maintenance of cellular integrity and adaptation to varying environmental conditions Ecological Significance: ​ Anaerobic Respiration and Biogeochemical Cycles ​ Decomposition of Organic Matter ​ Soil Health and Ecosystem Maintenance ​ Pollutant Depredation Obligate Anaerobes ​ Incapable of aerobic respiration ​ Variably tolerant of oxygen ​ Oxygen is toxic to them ​ Replicate at sites with low oxidation-reduction potential (Necrotic and Devascularized Tissue) ​ Obligate Anaerobes that commonly cause infection can tolerate atmospheric for 8 - 72 hours ​ Major components of the normal microflora on mucous membranes, especially of the oral cavity (Gingival, odontogenic, and pharyngeal) and lower GI tract ○​ Can cause disease when normal mucosal barriers break down Categories Based on Their Oxygen Tolerance: ​ Strict: ≤ 0.5% oxygen ​ Moderate: 2 to 8% oxygen ​ Aerotolerant Anaerobes: Tolerate atmospheric oxygen for a limited time Aerotolerant Anaerobes ​ Generates ATP through fermentation ​ Does not consume oxygen, but can defend themselves against reactive oxygen molecules ​ Thrives in environments that may contain oxygen, but do not rely on them ​ Classified based on their biochemical characteristics of environmental preferences ​ Lactobacillus, Streptococcus, and Bifidobacterium: Boost immunity and used as probiotics for gastrointestinal health ​ Propionibacteria (Including P. acidipropionici): Support skin health by maintaining microbial balance (Can also contribute to acne) ​ Generates propionic acid and vitamin B12 Aerotolerant Anaerobes Serve an Important Role: ​ Health ​ Environment ​ Industry ​ Food Sector Glycolysis ​ Provides electrons and energy ​ Produces 4 units of ATP, but 2 units of ATP are used in the process ​ Glucose is broken down into 2 units of pyruvate ​ 2 units of NAD+ is reduced to form 2 units of NADH by accepting electrons from glucose Pyruvate Processing or Pyruvate Reduction ​ Contributes electrons from NADH to the electron transport chain ​ Produces NADH and FADH2  ​ Pyruvate is used as an electron acceptor to oxidize NADH back to NAD+ to keep glycolysis running Electron Transport Chain ​ Uses alternative terminal electron acceptors instead of oxygen ​ Generates Proton Motive Force (Creates a proton gradient essential for ATP production Proton Motive Force ​ Drives ATP synthesis ​ Has lower energy yield compared to aerobic respiration Terminal Electron Acceptors ​ Receives electrons at the end of the electron transport chain ​ Allows the transfer of energy ​ Maintains the flow of electrons in aerobic conditions Common Terminal Electron Acceptors Terminal Electron Process Reduced Products Organisms Acceptors Nitrate (NO3-) Denitrification N2, N2O Paracoccus, Pseudomonas Sulfate (SO4²-) Sulfate Reduction H2S Desulfovibrio, Desulfotomaculum Carbon Dioxide Methanogenesis CH4 Methanogens (CO2) Acetogenesis Acetate Acetobacterium Metals (Fe³+) Metal Reduction Fe²+ Pseudomonas, Bacillus, Geobacter Fumarate Fumarate Reduction Succinate Wolinella Denitrification ​ Multi-step process that reduces nitrate (NO3-) to nitrogen gas (N2) ​ Paracoccus denitrificans ​ Pseudomonas aeruginosa Process of Denitrification: ​ NO3- > Nitrate Reductase > NO2- > Nitrite Reductase > NO > Nitric Oxide Reductase > N2O > Nitrous Oxide Reductase > N2 Sulfate Reductase  Sulfate Reduction Electron Electron Donor Chemical Examples Acceptor Reaction SO4²- Organotrophic 2CH3CHOHCO 2CH3CHOHCO Desulfovibrio Reducers O- (Lactate) O- + SO4²- > vulgaris 2CH3COO- + (Gram-negative) H2S + 2CO2 + 2H2O CH3COCOO- CH3COCOO- + (Pyruvate) SO4²- + 2H+ > CH3COCOO- + CO2 + H2S Lithotrophic H2 (Hydrogen 4H2 + SO4²- + Desulfovibrio Reducers Gas) 2H+ > H2S + (Gram-negative) 4H2O , Desulfotomaculu m (Gram-positive) Process of Sulfate Reductase: Methanogenesis ​ Process that reduces carbon dioxide (CO2) > Methane (CH4) Acetogenesis ​ Process that reduces carbon dioxide (CO2) > Acetate  Methanogenesis Acetogenesis Electron Acceptor CO2 (Carbon Dioxide) Electron Donor Lithotrophic Reducers H2 (Hydrogen Gas) Chemical Reaction CO2 + 4H2 > CH4 + 2H2O 4H2 + H+ + 2HCO³- > C2H3O²- + 4H2O Examples Methanogens Acetobacterium Paracoccus denitrificans ​ Facultative Anaerobes ​ Complete Denitrification ​ Nitrate (NO3-), Nitrite (NO2-), Nitric Oxide (NO), Nitrous Oxide (N2O) ​ Nitrogen gas (N2) ​ Moderate Yield Energy Escherichia coli ​ Facultative Anaerobes ​ Partial nitrate reduction or fumarate reduction ​ Nitrate (NO3-) Fumarate, other organic compounds ​ Nitrate (NO2-) or succinate ​ Low Yield Energy Importance of Anaerobic Respiration in Ecosystem ​ Living Organisms ○​ Humans ○​ Animals and Plants ○​ Bacteria ​ Environment ○​ Global cycles ○​ Survival ○​ Energy production ​ Food ○​ Bread ○​ Alcohol drinks ○​ Cheese Fermentation ​ Anaerobic metabolic process ​ Sugars > Energy, Alcohol, Carbon Dioxide, or Lactic Acid ​ Doesn't use oxygen History of Fermentation ​ One of the oldest microbiological processes ​ Accidentally discovered by Summerians and Egyptians (Wild yeast > Wine and Beer) ​ Over 2,000 years ago, Chinese invented fermented soy products like tofu and soy sauce 10,000 BC ​ Began in Northern Africart ​ Milk naturally fermented to yoghurt-like and cheese-like foods providing high-calorie sustenance 7,000 BC ​ Earliest form of alcoholic fermentation came from Jinhu (China) ​ Fruits, rice, and honey > Alcoholic Beverages 6000 BC ​ Winemaking originated from Georgia (Caucasus) ​ Roman colonization led to development of winemaking throughout the Mediterranean 500 BC ​ Chinese discovered the medicinal benefits of fermented soybean products ○​ Treats infections before modern antibiotics 1856 ​ Louis Pasteur proved microbes drive fermentation and spoilage ​ Pasteurization Today ​ Microbial fermentation produces complex pharmaceutical compounds using advanced bioreactors and recombinant technology (Vaccines and antibiotics) Types of Fermentation ​ Lactic Acid Fermentation ○​ Glucose > Lactic Acid ○​ Lactobacillus and muscle cells during intense exercise ○​ Allows ATP production when energy levels are low (Muscle Fatigue) ○​ Two types of Lactic Acid Fermentation: ​ Homolactic Fermentation ​ Heterolactic Fermentation ○​ Crucial in food preservation and production Process of Lactic Acid Fermentation: ​ Alcoholic Fermentation (Ethanol Fermentation) ○​ Glucose > Ethanol and Carbon Dioxide (Via Yeast) ○​ Streptomyces cerevisiae ○​ Two main steps: ​ Glycolysis ​ Fermentation ○​ Vital in making alcoholic beverages and in baking Process in Alcoholic Fermentation ​ Acetic Acid Fermentation ○​ Ethanol > Acetic acid ○​ Two main steps: ​ Alcoholic Fermentation ​ Acetic Acid Fermentation ○​ Oxidized by Acetic Acid Bacteria (AAB) (Acetobacter) ○​ Essential for vinegar production, enhancing food preservation, and flavor ○​ Pharmaceuticals and cleaning products Process in Acetic Acid Fermentation: ​ Butyric Acid Fermentation ○​ Mostly performed by anaerobic bacteria ○​ Main steps: ​ Glycolysis ​ Pyruvate Decarboxylation ​ Acetyl CoA Conversion ○​ Takes place in human colon ○​ Utilized by bioplastics, food, and chemical fields for flavoring ○​ By optimizing production parameters (pH and Temperature), batch or fed-batch fermentation techniques can improve the process (Normally produces 3 ATP per glucose Process in Butyric Acid Fermentation: Microbial Diversity ​ Fermentation ○​ Bacteria ​ Lactic Acid Bacteria ​ Lactobacillus, Lactococcus, Pediococcus ​ Acidification process enhances texture and flavor ​ Yogurt and cheese ​ Bacillus Species  ​ Bacillus subtilis ​ Key in alkaline fermentations (Asia and Africa) ​ Legume-based fermented foods ○​ Yeast ​ Saccharomyces, Candida ​ Breakdown of sugars > Alcohol and CO2 ​ Alcoholic beverages ○​ Mold ​ Aspergillus, Rhizopus ​ Enzyme production > Breakdown of complex carbohydrates ​ Soy-based foods Enzymatic Processes ​ Enzymes in fermentation act as biological catalyst ​ Speeds up chemical reactions (Sugars > Energy, Alcohol, or other byproducts) ​ Enzymes are nature's factory workers Glycolytic Enzymes ​ During glycolysis, glucose is broken down to two molecules of pyruvate through series of enzyme-driven reactions ​ 2 ATP molecules and 2 NADH molecules (later used or recycled depending on type of fermentation ​ Hexokinase and phosphofructokinase Pyruvate Decarboxylase ​ Enzyme that catalyzes conversion of pyruvate to acetaldehyde and carbon dioxide ​ Facilitates removal of carboxyl group from pyruvate in the first step of ethanol production ​ Starts alcohol fermentation ​ Pyruvate > Pyruvate Decarboxylase > Acetaldehyde Alcohol Dehydrogenase ​ Catalyzes the reduction of acetaldehyde to ethanol by transferring electrons from NADH to acetaldehyde ​ Regenerates NAD+ and completing the process of alcoholic fermentation ​ Ethanol > Alcohol Dehydrogenase > Acetaldehyde Lactate Dehydrogenase ​ Catalyzes conversion of pyruvate to lactate by transferring electrons from NADH to pyruvate ​ Regenerates NAD+ ​ Enables glycolysis to continue under anaerobic conditions during lactic acid fermentation  ​ Pyruvate > Lactate Dehydrogenase > Lactate Application of Fermentation ​ Food Production ○​ Alcoholic beverages ○​ Dairy products ○​ Bread making ○​ Pickling ○​ Fermented foods ​ Pharmaceuticals ○​ Antibiotics ○​ Vaccines and biologics ○​ Enzyme production ​ Biofuels ○​ Ethanol ○​ Butanol ○​ Biogas ○​ Biodiesel Regulation of Metabolic Pathways ​ Enzyme Regulation ○​ Allosteric effects ○​ Phosphorylation ○​ Feedback inhibition ​ Gene Expression ○​ Transcription factors regulate gene expression in response to environmental circumstances ​ Metabolite Flux ○​ Pathway flow controlled by enzyme activity and substrate availability to direct resources to desired products Optimization Strategies ​ Genetic Engineering ○​ Introducing or altering pathways ○​ Eliminates competing paths ○​ Direct resources to desired paths ​ Metabolic Flux Analysis ○​ Identifies limitations ○​ Improves metabolite flow to increase yields ​ Adaptive and Directed Evolution ○​ Putting strains under stress and adding mutations improves performance ​ Process of Optimization ○​ Fed-batch cultures, oxygen, pH control, and medium modifications improve efficiency while reducing byproducts  ​ Systems Biology and AI ○​ Optimizing strains, pathways, and circumstances using omics data and machine learning Challenges and Future Directions ​ Carbon Efficiency ○​ Efficient utilization of carbon sources (Glucose) remains a key difficulty, especially in industrial-scale fermentation that requires vast amounts of it ​ Byproduct Creation ○​ Reducing undesired byproducts (Acetic Acid or other organic acids) is crucial for increasing total yield of target product ​ Strain Stability ○​ Engineered strains may be unstable, resulting in loss of desirable features over time ○​ Continuous monitoring it and stabilizing strategies are required to maintain performance in large-scale fermentation ​ Scalability ○​ Differences in nutrient dynamics, mixing, and oxygen transfer can make it challenging to translate laboratory-scale results to industrial-scale fermentation Environmental Impact ​ Precision Fermentation in Protein Production ○​ Uses bacteria, yeast, and fungi to create food ingredients (Proteins in large bioreactors) ○​ Products are called fermentation produced proteins ○​ Used in many food ingredients ​ Reduction in Greenhouse Gas Emissions ○​ Greener alternative to traditional farming ○​ Livestock farming = 14.5% global greenhouse gas emissions ○​ Fermentation avoids this, offering a more sustainable solution ​ Land and Water Conservation ○​ Livestock farming uses 70% of agricultural land and requires huge amounts of water ○​ Precision fermentation uses much less land and water ○​ Reduces environmental footprint by up to 90% ​ Reduction of Biodiversity loss ○​ Livestock farming leads to deforestation and less wildlife habitats ○​ Precision fermentation reduces the need for land, helping conserve forests and protect ecosystems PHOTOSYNTHESIS Phototrophy ​ Light eating ​ Metabolic process where organisms use light energy into chemical energy (ATP) ​ Broadly classified into: ○​ Photoautotrophs- Uses light energy to fix carbon dioxide into organic compounds (Plants, Algae, Cyanobacteria) ○​ Heteroautotrophs- Uses light but rely on organic compounds as a carbon sources (Purple Bacteria) Photosynthesis ​ Process that both convert sunlight into ATP ​ Uses ATP to carbon dioxide into organic compounds ​ Classified into: ○​ Oxygenic Photosynthesis- Organism that consume H2O and produce O2 as a waste product ○​ Anoxygenic Photosynthesis- Photosynthetic organisms that do not produce O2 Chlorophyll and Bacteriochlorophyll ​ Light-absorbing pigments essential for photosynthesis ​ Tetrapyrroles (4 pyrrole rings: Five membered heterocyclic rings) ​ Contains specific substituents and hydrophobic alcohol (phytol) that helps them anchor into photosynthetic membranes Chlorophyll Bacteriochlorophyll Found in plants, algae, and cyanobacteria Found in photosynthetic bacteria Captures light energy for oxygenic Captures light energy for anoxygenic photosynthesis (Produces oxygen) photosynthesis (Does not produce oxygen) Chlorophyll A (Main pigment), B, C, D, F Bacteriochlorophyll A, B, C, D, E, F, G (Accessory pigment) 680-430 nm 800-925 nm H2O is the electron donor Uses other electron donor like H2S or other organic compounds Reaction Centers ​ These are the specialized protein-pigment complexes responsible for converting light energy into chemical energy ​ Chlorophyll and Bacteriochlorophyll molecules are attached to proteins and housed within membranes to form photo complexes Mechanism of Energy Conversion in RC  ​ Photocomplexes- Protein-pigment assemblies crucial for photosynthesis ○​ Three measures: ​ Photosystem I (PSI) and Photosystem II (PSII) ​ Light-Harvesting Complexes (LHC) ​ Electron Transport Chain (ETC) ○​ 50-300 chlorophyll/Bchl molecules are part of these complexes Light-Harvesting Complexes and Antenna Pigments ​ Photosynthetic reaction centers are surrounded by more light-harvesting chlorophylls/bacteriochlorophylls ​ Antenna Pigments- Function to maximize light absorption and direct energy to the reaction center Antenna Pigments and Light Harvesting ​ Light energy is absorbed by light harvesting molecules ​ It's then transferred to the RC where photosynthetic electron transport reactions begin ​ Pigment molecules are secured within the membrane by specific pigment-binding proteins ​ At low light intensities, this arrangement for concentrating energy allows RCs to receive light energy that would otherwise be missed Photosynthetic Membranes in Eukaryotes ​ In eukaryotes, photosynthesis occurs in chloroplasts ​ Chlorophyll pigments and all the other components of the light-gathering apparatus exist within membranes in the cell ​ Location of photosynthetic membranes differs between prokaryotic and eukaryotic phototrophs ​ Chloroplast Structure: ○​ Thylakoid membranes ​ Site of light reactions ​ House PSI, PSII, and ATP synthase ○​ Grana ​ Stacked structures of thylakoids ​ Optimizes the surface area for light absorption ​ Chloroplast is divided into two regions: ○​ Stroma- Fluid-filled region surrounding thylakoids where the Calvin cycle occurs ○​ Lumen- Space within thylakoids where protons are pumped during the light reactions to create a proton gradient Thylakoid Membranes and Energy Generation ​ Thylakoid membranes play a crucial role in light energy conversion: ○​ The light-driven proton motive force (PMF) created by the ETC drives ATP synthesis ​ Proton gradient across the thylakoid membrane:  ○​ Protons are pumped into the lumen, generating a gradient ○​ ATP synthase uses this gradient to produce ATP in the stroma Photosynthetic Membranes in Prokaryotes ​ Prokaryotic Photosynthesis ○​ Occurs in specialized internal membrane systems ○​ Lack chloroplast ​ Membrane Invaginations in the cytoplasmic membrane form chromatophores and lamellae ○​ Chromatophores- Membrane vesicles containing photosynthetic pigments ○​ Lamellae- Stacked membrane structures that provide a large surface area for light absorption Lamellar Membranes in Cyanobacteria ​ Cyanobacteria- Photosynthetic bacteria that resemble plant chloroplast ​ Lamellar Membranes- Internal membrane structures that are analogous to thylakoids in plant chloroplasts ○​ This membrane stacks the photosynthetic pigments and RCs for photosynthesis ○​ Internal membrane systems that help cyanobacteria capture light energy and convert it into chemical energy Chlorosomes in Anoxygenic Phototrophs ​ Chlorosomes- Specialized light-harvesting structures in green sulfur bacteria and green non-sulfur bacteria ○​ Function: Captures light in low light environments (Deep aquatic habitats) ○​ Structure: Bacteriochlorophyll (Bchl) molecules are densely packed in a crystalline array, forming an efficient light-harvesting complex ​ Unlike other systems, Bchl in chlorosomes is not attached to proteins but arranged in dense arrays ○​ Energy absorbed by chlorosomes is transferred to the RC via Fenna-Matthews-Olson complex (FMO) proteins ​ Dense packing of Bchl c, d, or e in chlorosomes allows efficient absorption of light, especially in low-light environments, arranged in crystalline-like structures along chlorosome’s long axis ​ The absorption of light energy is funneled through the pigments toward the baseplate, a protein structure at the bottom of the chlorosome ​ From the baseplate, energy is passed to Bchl a (Located in the RC within the cytoplasmic membrane through the small protein called FMO protein) ​ FMO protein connects the chlorosome to the RC, ensuring efficient energy transfer with minimal loss. ○​ This protein is important for bridging the gap between the light-harvesting pigments in the chlorosome and the photosynthetic RC Carotenoids and Phycobilins  ​ Carotenoids ○​ Help plants in capturing and utilizing light energy ○​ Absorbs light in the blue-green and violet region of the spectrum ○​ Most widespread accessory pigments in phototrophs ○​ Function as photoprotective agents ○​ Usually found in Cyanobacteria, algae, some species of Archaea, fungi and plant ​ Phycobiliproteins ○​ Proteins that capture light energy for photosynthesis in algae and cyanobacteria ○​ Contains red or blue-green linear tetrapyrroles called billins ○​ They cluster into structures called phycobilisomes ​ Phycobilisomes are attached to the thylakoid membranes of cyanobacteria to facilitate the light energy transfer to chlorophyll ○​ Three types of phycobiliproteins: ​ Phycocyanin- 620 nm ​ Phycoerythrin- 550 nm ​ Allophycocyanin- 650 nm ​ Allophycocyanin are in direct contact with the RC chlorophyll ​ Allophycocyanin is surrounded by phycocyanin, phycoerythrin, or both Anoxygenic Photosynthesis ​ Process where light energy is captured and converted into chemical energy without the production of oxygen ​ Relies on various donors such as hydrogen sulfide, sulfur, or organic compounds ​ Key characteristics: ○​ Electron Donor- Water is not used (Hydrogen sulfide, sulfur, or organic compounds is used) ○​ Oxygen Production- Oxygen is not produced ○​ Photosystem Involvement- Primarily involves Photosystem I (Captures longer wavelength) ○​ Organisms- Primarily bacteria (Purple sulfur bacteria: Thiospirillum, Purple non-sulfur bacteria: Rhodobacter, Green sulfur bacteria: Chlorobium, and Green non-sulfur bacteria: Chloroflexus ​ Electron Transport Chain- Arranged in a photosynthetic membrane in order of their increasingly more electro-positive reduction potential (E0’) ​ Photosynthetic Reaction Center- Complex macromolecular structures localized within photosynthetic membranes (Sunlight > Energy > Food for Plant) Anoxygenic Photosynthesis of Purple Bacteria ​ One of the microorganisms that undergo anoxygenic photosynthesis for metabolism (Q-type RC with Quinone carrier molecule) ​ Components of Q-type reaction center: ○​ P870- Special pair of Bacteriochlorophyll that absorbs light energy (870 nm) ○​ Bacteriopheophytin A (BPh)- Intermediate electron carrier within the RC ○​ Quinone (Q)- A small carrier of electrons  ○​ Cytochrome (Cyt)- Protein that carries electron and contributes to Proton Motive Force ​ Anoxygenic photosynthesis begins when the light energy excites the P870 ○​ Increases its Redox potential or electro-positive reaction potential from 0.5 volts to 1.0 volts ​ Excited electrons are transferred from P870 to carrier molecules ○​ The transfer generates a Proton Motive Force that synthesizes ATP ○​ Anoxygenic electron donor, H2S, drives to generate NADPH ​ Electron cycle back to P870 ○​ Cyclic Photophosphorylation- Closed loop of electron Overview of Electron Flow in Anoxygenic Phototrophs Photosynthetic Reaction Centers Type 1 (FeS type) Type 2 (Pheo-Q type) Bacteriochlorophyll and chlorophyll a (Green Bacteriochlorophyll Sulfur Bacteria) Hydroxychlorophyll a (Heliobacteria) Purple Bacteria and Cyanobacteria Photosystem I (PSI) Photosystem II (PSII) Anoxygenic Phototrophs Pathways 1.​ Oxygen is not released because P879 of PSII is not present a.​ Water is too electro-positive to act as the electron donor for the photosystem 2.​ Depending on the species, the RC can consist of chlorophyll, bacteriochlorophyll, or other pigments a.​ The RC in purple bacteria is called P870 3.​ Some of the carriers within the electron chain are different, including bacteriopheophytin  a.​ Bacteriopheophytin- Bacteriochlorophyll without it's Mg²+ ion 4.​ P870 is a poor electron donor in its ground state a.​ When light or infrared light is present, it turns the weak or poor electron donor into a strong electron donor (870) b.​ It will go into Bacteriochlorophyll (Bchl), then BPh into QA, QB, Q pool, Cyt BCl, Cyt C2, back to P870 5.​ Electrons cycle back to reduce P870, so this is a cycle electron transport chain leading to generation of ATP through cyclic photophosphorylation 6.​ Unlike in oxygenic photosynthesis, where NADPH is the terminal electron acceptor, no NADPH is made because electrons are cycling back into the system ​ Photosystem a.​ Anoxygenic Phototrophs typically have a single photosystem (PSI or PSII) b.​ Some species like purple sulfur bacteria may use both PSI and PSII, but oxygen is not produced ​ Electron Donors a.​ Hydrogen Sulfide (H2S) b.​ Elemental Sulfur (S) c.​ Organic compounds like Acetate d.​ Ferrous iron (Fe²+) e.​ Reduces photosystem's RC ​ Electron Transport Chain in purple sulfur bacteria: a.​ The electron donor (H2S) is oxidized to sulfur (S) and the electrons are transferred to a RC, typically P870 (Purple Bacteria) b.​ The electrons are passed through the electron transport chain, which involves proteins like cytochrome c and quinones c.​ ATP is generated via cyclic photophosphorylation (where electrons cycle back to the reaction center to generate more ATP) d.​ Electrons may reduce NAD+ to NADH (or NADPH), which is used for carbon fixation or other metabolic pathways ​ Electron Transport Chain in purple nonsulfur bacteria: a.​ These organisms can use a variety of electron donors (such as organic compounds) b.​ Electron Flow follows similar patterns (Electrons > RC > NADPH) ​ Energy Production: Electron > Proton Gradient ATP via ATP Synthase a.​ Similar to chemiosmotic gradient used in cellular respiration, where PMF is harnessed to produce ATP ​ Reduction of NAD(P)H: Reduce NAD+ or NADP+ to NADH or NADPH a.​ Essential for biosynthetic processes like carbon fixation ​ Final Electron Acceptor NAD, Quinones Green Sulfur Bacteria Purple Bacteria FeS Cluster Quinone FeS type RC that donate electrons directly to Q-type RC donates electron to a Quinone, low potential FeS-proteins to reduce however this quinones (E0’ about 0 V) are ferredoxin insufficiently electronegative to provide the reducing power (E0’ of -0.32 V for NAD(P)H and -0.42 V for ferredoxin) needed for CO2 fixation and other biosynthetic reactions Uses Fdred when fixing CO2 using rTCA Q-type RCs produce electrons that are cycle insufficiently electronegative to reduce NAD(P)+, these phototrophs require reverse electron transport to produce NAD(P)H they need to carry out CO2 fixation and other biosynthetic reactions Electrons from ferredoxin can also pass to Reducing power is needed for biosynthetic ferredoxin-NAD+ oxidoreductase for the reactions for growth production of NAD(P)H needed for biosynthesis Unsure cyclic and non-cyclic phosphorylation. Exemplify cycle phosphorylation for ATP Proposed that the FeS type RC in these production as long as light is present, phototrophs can transfer electrons directly to however reducing power is needed for menaquinone, generating a PMF resulting in biosynthetic reactions for growth cyclic photophosphorylation as seen in purple bacteria Non-cyclic phosphorylation electrons from external electron donors (H2S) enter at the level of the menaquinone pool. These electrons would be transferred through the RC and then to ferredoxin where they would ultimately be channeled into biosynthetic reactions without the need for reverse electron transport In Heliobacteria, FeS type phototrophs that are not autotrophic, cyclic photophosphorylation have also not been demonstrated Photoheterotrophic bacteria reducing power needs are less extensive than for photoautotrophs and thus it is more likely that cyclic flow occurs to generate sufficient ATP for biosynthesis Oxygenic Photosynthesis ​ Key characteristics: ○​ Electron Donor- Water ○​ Production of Oxygen- Oxygen as byproduct ○​ Photosynthetic RC- Both FeS-type and Q-type ○​ Photosystems: PSI or P700 and PSII or P680 ○​ “Z scheme” of Photosynthesis- Pathway of electron resembling the letter Z turned on its side ○​ Location- Eukaryotic cells: Chloroplast, Cyanobacteria: Cytoplasm Electron Flow and ATP Synthesis in OP ​ PSII is activated by protons ○​ This leads to the oxidation of H2O at the Mn4Ca cluster within the water- oxidizing complex ○​ This process results in the production of O2 and 4H+ ​ Each electron transferred to pheophytin moves through a series of quinones (QA and QB) within PSII and eventually reaches the plastoquinone pool (PQ/PQH2) ​ Electrons from PQH2 are passed through cytochrome b6f and then through plastocyanin (copper containing protein) before being delivered to the PSI reaction center ○​ For every two water molecules oxidized into one O2, a total of 12 protons are released into the lumen to power ATP synthase ​ The absorption of light by P700 in PSI enables it to accept electrons donated by plastocyanin ○​ These electrons pass through several intermediates within PSI, ultimately resulting in the reaction of NADP+ to NADPH ​ For each water molecule split by PSII, two protons are produced and four protons are transported across the membrane for every pair of electrons passing through the ETC ○​ This leads to a total of 12 protons being translocated for every O2 molecule generated  ○​ ATP synthase then uses this PMF to synthesize ATP ​ Oxygenic photosynthesis leads to non-cyclic phosphorylation because the electrons do not return to reduce the oxidized P680 ○​ Instead they are used to reduce NADP+ ​ When the cell needs less NADPH for biosynthesis, oxygenic phototrophs can switch to cyclic photophosphorylation The Light-Independent Reactions: ​ Calvin-Benson Cycle ○​ Responsible for CO2 fixation ○​ Found in cytoplasm of photosynthetic bacteria and the stroma of chloroplasts in eukaryotes ○​ Light-dependent reactions are divided into three main stages: ​ Fixation- Ribulose bisphosphate carboxylase (RuBisCO) catalyzes the addition of CO2 to ribulose bisphosphate (RuBP), producing 3-phosphoglycerate (3-PGA) ​ Reduction- Six molecules of both ATP and NADPH (from the light-dependent reactions are utilized to convert 3-PGA into G3P ​ Portion of the G3P is used to form glucose ​ Regeneration- G3P not involved in glucose synthesis is used to regenerate RuBP, ensuring the continuation of CO2 ​ This process requires three additional ATP molecules Anoxygenic Photosynthesis in Oxygenic Phototrophs ​ Photosystem 1 and 2 usually work together in oxygenic photosynthesis ○​ Some organisms can still carry out photosynthesis using only PS1 if PS2 is blocked ○​ This leads to cyclic photophosphorylation and the use of alternative sources of reducing power for CO2 Reduction resulting in anoxygenic photosynthesis in oxygenic phototrophs ​ Some cyanobacteria can use H2S as an electron donor ○​ Producing sulfur granules outside their cells similar to those made by green sulfur bacteria Microbial Genetics Molecular Information Flow and Protein Processing ​ Molecular Biology and Genetic Elements ○​ Gene- Functional unit of genetic information ○​ Genomes- Total genetic elements in an individual ○​ DNA and RNA- Polynucleotide made of informational macromolecules ​ Properties of Double Helix:  ○​ Complimentary ○​ Antiparallel ○​ Minor and major grooves ​ Nucleoside- Sugar + Base ​ Nucleotide- Sugar, Base, and phosphate ​ Pyrimidines- Single ring with one carbon (Cytosine and Thymine) ​ Purines- Two fused rings with five carbons and four nitrogens (Guanine and Adenine) ​ Size, Shape, and Supercooling ○​ Nucleotide Base Pairs- DNA size unit measure ​ 1000 bp = 1 kbp ​ E. coli = 4640 kbp ○​ Bacteria and Archaea undergo Supercoiling via ds breaks ​ Topoisomerase or DNA gyrase ​ Can be + or - ​ + Supercoiling- Overwound ​ - Supercoiling- Underwound ​ Multiple supercoiling > Supercoiled Domains ○​ Eukaryotes wound their DNA around proteins ​ Genes and the Steps in Biological Information Flow ○​ Central Dogma ​ Replication ​ Transcription ​ Translation ○​ Eukaryotes: ​ One gene ​ One mRNA ○​ Prokaryotes: ​ One mRNA ​ Several different proteins ○​ Genetic Elements- Contains the genetic material ​ Plasmid ​ Transposable Elements Chromosomal Gene Arrangement ​ E. coli ○​ 4,639 Mb had been mapped out ○​ Mostly composed of protein-coding genes ○​ Some genes form operons but majority are found as a single gene ​ Eukaryotes ○​ Intervening sequences or repetitive sequence Plasmids ​ Common in prokaryotes ​ dsDNA  ​ Free DNA in cytoplasm ​ Circular or linear ​ Varies in size ​ Exist in different copy numbers ​ Uses the same replication enzyme used by the chromosome ​ R Plasmid- Inactivate antibiotics or protects the cell ​ Pathogenicity ○​ Bacterioricins ​ Nitrogen- Fixing nodules of Rhizobium ​ Depredation of hydrocarbons or toxic pollutants, such as poly chlorinated biphenyls, herbicides, pesticides ​ HGT DNA Replication ​ Bidirectional ​ Semiconservative ​ Proceeds in 5’ - 3’ direction ​ Enzymes ○​ DNA Pol I-V ○​ Primase Major Enzymes that Participate in DNA Replication in Bacteria ​ DNA Gyrase (gyrAB)- Replaces supercoils ahead of replisome ​ Origins Bonding Protein (dnaA)- Binds origin of replication to open double helix ​ Helicase Loader (dnaC)- Loads helicase at origin ​ Helicase (dnaB)- Unwinds double helix at replication fork ​ Single-Strand Binding Protein (ssb)- Prevents single strands from annealing ​ Primase (dnaG)- Primes new strands of DNA ​ DNA Polymerase III- Main polymerizing enzyme ​ Sliding Clamp (dnaN)- Holds Pol III on DNA ​ Clamp Loader (holA-E)- Loads Pol III onto sliding Clamp ​ Dimerization Subunit (dnaX)- Holds together the two core enzymes for the leading and lagging strands ​ Polymerase Subunit (dnaE)- Strand elongation ​ Proofreading Subunit (dnaQ)- Proofreading ​ DNA Polymerase I (polA)- Excises RNA primer and fills in gap ​ DNA Ligase (ligA, ligB)- Seals nicks in DNA ​ Tus Protein (tus)- Binds terminus and blocks progress of the replication fork ​ Topoisomerase IV (parCE)- Unlinking of interlocked circles DNA Replication Process: ​ Initiation ○​ Origin-binding protein ○​ Helicase and loader  ○​ SSBP ○​ Primase ○​ DNA Pol III ​ Elongation ○​ Leading Strand ○​ Lagging Strand ​ Okizaki Fragments ​ Termination ○​ DNA Pol I ○​ DNA Ligase In Context of Bacteria and Archaea ​ Circular ​ Theta Structure ​ Replisome- complex of enzymes ○​ Present in both ○​ Looping out ○​ Primosome- Helicase and primase ○​ Tus Proteins- Recognizes Ter sites ○​ FtsZ Proteins- Separator DNA into daughter cells ○​ Proofreading ​ DNA Pol ​ DNA Pol III inserts appropriate base ​ DNA Pol I and III has exonuclease activity Transcription ​ DNA > RNA: mRNA, tRNA, rRNA ○​ Genetic Level: mRNA ○​ Structural: tRNA and rRNA ​ DNA vs RNA ○​ DNA = Double-stranded ○​ RNA = Single-stranded ​ Primary Structure- Unfolded (mRNA) ​ Degraded by ribonuclease ​ More rapid turnover rate ​ Secondary Structure ​ Long-lived ​ Bacteria ○​ Only a segment of DNA is transcribed ○​ RNA Pol- Simple ​ 5 Subunits: ß, ß’, α, Ω, and σ ​ α is in two copies ​ RNA Pol- Core Enzymes: ß, ß’, α, and Ω ​ Sigma Recognizes Promoters  ○​ Specific for a group of genes ○​ A mechanism for gene regulation ​ RNA Pol starts the process of transcription ○​ Transcription bubble ​ E. coli σ70 ○​ Recognizes two highly conserved regions within the promoter: -10 Region (TATAAT) and -35 Region (TTGACA) ​ Allows them to recognize different promoter sequences ​ Pattern ​ Strong Promoters- Sequences that conform the most to the consensus sequences ​ Transcriptional Units ​ Both coding and non-coding proteins undergo processing ​ Operon ○​ One RNA > Several different proteins ○​ Polycistronic mRNA with multiple open reading frames ○​ Coordinated expression ​ Regulations ​ Termination ○​ Rho-dependent ​ Rho-protein ​ Binds to mRNA ○​ Rho-independent ​ Stem-loop or hairpin loop followed by a run of A ​ Will yield many U, which has a strong termination sequence ​ Archaea and Eukarya ○​ Archaea- One RNA Pol- with 11-13 subunits ○​ Eukarya- Three RNA Pol ○​ Promoters- 6-8 bp of ‘TATA’ box ​ TATA Binding Proteins (TBP) ​ TF ○​ B Recognition Element (BRE) Sequences ​ Transcription Factor B (TFB) ​ Upstream of TATA Box ​ Archaean Termination Hypothesis ○​ Repeated runs of T ○​ Euryarchaeota: Eta protein ​ RNA Processing in Eukaryotes and Intervening Sequences in Archaea ​ Primary Transcript ​ Process: ○​ Splicing of Intervening Sequences ​ Spliceosome  ​ Ribonuclease ○​ Capping ​ Methylated guanine nucleotide ○​ Polyadenylation ​ Poly-A-Tail Translation ​ Protein Structure ​ tRNA ​ Translation and the Genetic Code Genetic Code as Expressed by Triplet Base Sequences of mRNA Codon Amino Acid Codo Amino Codon Amino Acid Codon Amino Acid n Acid UUU Phenylalanine UCU Serine UAU Tyrosine UGU Cysteine UUC Phenylalanine UCC Serine UAC Tyrosine UGC Cysteine UUA Leucine UCA Serine UAA None (stop UGA None (stop signal) signal) UUG Leucine UCG Serine UAG None (stop UGG Tryptophan signal) CUU Leucine CCU Proline CAU Histidine CGU Arginine CUC Leucine CCC Proline CAC Histidine CGC Arginine CUA Leucine CCA Proline CAA Glutamine CGA Arginine CUG Leucine CCG Proline CAG Glutamine CGG Arginine AUU Isoleucine ACU Threonine AAU Asparagine AGU Serine AUC Isoleucine ACC Threonine AAC Asparagine AGC Serine AUA Isoleucine ACA Threonine AAA Lysine AGA Arginine AUG Methionine ACG Threonine AAG Lysine AGG Arginine (start) GUU Valine GCU Alanine GAU Aspartic GGU Glycine Acid GUC Valine GCC Alanine GAC Aspartic GGC Glycine Acid GUC Valine GCA Alanine GAA Glutamic GGA Glycine Acid GUG Valine GCG Alanine GAG Glutamic GGG Glycine Acid ​ Properties: ○​ Triplet ○​ Degenerate ​ Codon Bias ​ tRNA Specificity ○​ E. coli: ​ 6 tRNAs for Leu ​ 1 Lsyl tRNA recognizes 2 Lys codons ​ Wobble ​ Start and Stop Codons and Reading Frames ○​ Start Codon ​ Bacteria: fMet ​ Archeae and Eukarya: Met ○​ Stop Codons ​ Nonsense ​ In some cases, stop codons are hijacked, encoding Selenocysteine and Pyrrolysine ○​ Open Reading Frame (ORF) ​ Start codon-Number of codons-Stop codon ​ A slight shift can drastically affect the AA being expressed ​ Bacteria: AUG + Shine-Dalgarno Sequence/Ribosome-Binding Site (RBS) ○​ Start Codon: GUG ​ The Mechanism of Protein Synthesis ○​ mRNA, tRNA, ribosomes, other proteins, and guanosine triphosphate (GTP) ​ Ribosomes- A large dynamic complex of proteins, which synthesized proteins ​ Bacteria and Archaea: 30S + 50S = 70S ○​ 30S:16S rRNA and 21 proteins ○​ 50S: 5S and 23S rRNA, and 31 proteins ○​ Thus, 52 distinct proteins ○​ Bacteria ​ Initiation: ​ Free 30S + mRNA ​ Initiation Complex: 30S, mRNA, fMet tRNA, and IF ​ +50S ​ RBS: Secure Ribosome- mRNA complex ○​ Polycistronic mRNA has several RBS  ○​ Polysome- A complex where several ribosomes translate an mRNA at the same time ○​ Bacteria ​ Elongation ​ fMet tRNA at P site ​ EF-TU dissociates ○​ EF-Ts: Regenerate via GDP and GTP exchange ​ Translocation through EF-G, emptying the A site ​ Termination ​ Release Factors (RF) recognizes stop codon and cleave the polypeptide products ○​ Role of rRNA ​ Bacteria ​ Ribosome subunit association ​ 16S rRNA + RBS ​ Positioning tRNAs on the ribosome ○​ tRNA acceptor end + 23S rRNA ​ Peptidyl Transferase Reaction ○​ Happens in 50S and catalyzed solely by 23S rRNA ​ Translocation ○​ Trapped Ribosomes ​ Bacteria: Trans-translation ​ tmRNA: Ala tRNA and mRNA ○​ Has a stop codon ○​ Short stretch of mRNA serves as a signal for protease Protein Processing, Secretion, and Targeting ​ Assisted Protein Folding and Chaperons ○​ E. coli ​ dnaK and dnaJ ○​ Protein Secretion ​ Secretion (SEC) ​ Twin-Arginine Translocation (TAT) ○​ Protein Secretion: Gram-Negative Bacteria ​ Type I (E. coli bacteriocin) ​ Type II (V. cholerae glucanase exoenzymes) ​ Type III (Effector molecules of Chlamydia and Salmonella) ​ Type IV (Agrobacterium DNA) ​ Type V (E. coli and H. influenza adhesion proteins) ​ Type VI (P. aeruginosa toxin) ​ Type VII ​ Type VIII ​ Type IX Mutation ​ Wild-type-strain- A strain of organism isolated in nature ​ Mutant- The individual that carries the mutation in its genome ​ Naming System: ○​ Genotype: Three lowercase letters, followed by a capital letter (all in italics) ​ If mutant is isolated, a number is added ​ hisC, (hisC1, hisC2, and hisC3) for histidine ○​ Phenotype: Capital letter followed by two lowercase letters with + and - to indicate the presence of absence of that characteristic ​ His+ and His- (Histidine) ​ Molecular Basis ○​ Spontaneous- Occur naturally due to inherent errors in the process ○​ Induced- Caused by external agents called mutagens ​ Types: ○​ Point Mutation ​ Missense- Mutation that changes the amino acids that are incorporated into a protein ​ Nonsense- Mutation that introduces a stop codon into the genetic code and prevents the protein from being made completely ​ Silent- No change in amino acid sequence ​ Transition- Purine base is replaced with another purine, or pyrimidine with another pyrimidine ​ Transversion- Purine is replaced with pyrimidine, or vise versa ○​ Insertions or Deletion ​ Frameshift Mutation Gene Transfer VIRUS ​ Nonliving ​ Parasites ​ Composed of one type of nucleic acid, capsid, and/or lipid membrane ​ Baltimore Classifications: ○​ Class 1: dsDNA (Herpesviridae) ○​ Class 2: ssDNA (Parvoviridae) ○​ Class 3: dsRNA (Reoviridae) ○​ Class 4: +ssRNA (Coronaviridae) ○​ Class 5: -ssRNA (Rhabdoviridae) ○​ Class 6: +ssRNA with RT (Retroviridae) ○​ Class 7: dsDNA with RT (Hepadnaviridae) GENE CONTROL Operon ​ Lac Operon ○​ Components: ​ P- Promoter ​ I- Regulator Gene > mRNA > Regressor Protein ​ O- Operator ​ Z- B-galactosidase (Cleaves lactose to glucose and galactose) ​ Y- Lactose Permease (Translocate Lactose) ​ A- Transacetylase (Breaks sugar) ○​ Mechanism: ​ Lactose Absent: Operon OFF ​ Lactose Present: Operon ON