Microbial Ecology: MICR3213/BC31M Past Paper PDF

Microbial Ecology MICR3213/BC31M: Applied and Environmental Microbiology Dr. Stacy Stephenson-Clarke [email protected] Inspirational Quote of the Day Class Representative? MICR3213 Timetable MICR3213 Timetable Course Outline  Course Assessments ❑...

Microbial Ecology MICR3213/BC31M: Applied and Environmental Microbiology Dr. Stacy Stephenson-Clarke [email protected] Inspirational Quote of the Day Class Representative? MICR3213 Timetable MICR3213 Timetable Course Outline  Course Assessments ❑ Final Written Examination (2 hours): 60% ❑ Course Work: 40% ▪ 2 In-course Tests 20% ▪ Monday Oct 7 at 9:00 am ▪ Monday Nov 25 at 9:00 am ▪ Laboratory Reports 20% ▪ A practical section of 36 hours.  Learning Objectives: ❑Understand the ecology of microorganisms at population, community and ecosystem levels ❑Describe the approaches used to study microorganisms in their natural environments and the limitations associated with each ❑Describe community function and dynamics at both the molecular and the organismal level  Microbial Ecology: Further Reading ❑ See Madigan et al. (2020). Brock Biology of Microorganisms (16th Ed). Person Education Limited ❑Chapters 19 and 20  Lecture Objectives At the end of the lecture, students will be able to: ❑ Understand the ecology of microorganisms at population, community and ecosystem levels  Microbial Ecology Considerations ❑ Microbes and Ecosystem Niches ❑ Organization of Ecosystems ❑ Role of Microbes in Biogeochemical Cycling ❑ Microbial Environments and Microenvironments  Microbial Ecology ❑ Study of inter-relationships between microorganisms and their environments ECOSYSTEM COMMUNITY GUILD POPULATION INDIVIDUAL  General Ecological Concepts ❑Ecosystem The sum total of all organisms and abiotic factors in a particular environment ❑Habitat Portion of an ecosystem where a community could reside  General Ecological Concepts ❑Population a group of microorganisms of the same species that reside in the same place at the same time may be descendants of a single cell ❑Community a community consists of populations living in association with other populations  General Ecological Concepts Ecosystem Service: Biogeochemistry and Nutrient Cycles ❑Guilds metabolically related microbial populations sets of guilds form microbial communities that interact with macroorganisms and abiotic factors in the ecosystem ❑Niche habitat shared by a guild supplies nutrients as well as conditions for growth Microbial Ecology  General Ecological Concepts Diversity of microbial species in an ecosystem is expressed in two ways ❑species richness: total number of different species present ❑species abundance: proportion of each species in an ecosystem Microbial species richness and abundance are functions of the kinds and amounts of nutrients available in each habitat Energetics and carbon heterotroph flow in microbial metabolism (e.g., NH4+, S, H2S, Fe2+) autotroph  Objectives in Microbial Ecology ❑ Understanding the biodiversity of microorganisms in nature, and interactions in communities ❑ Measurement of microbial activities in nature, and monitoring of effects on ecosystems ❑ Activities commonly measured when studying microorganisms within an ecosystem: ❑ Primary production of organic matter (phototrophic, chemolithotrophic activity) ❑ CO2 + H2O + energy → new biomass ❑ Decomposition of organic matter (chemoorganotrophic/heterotrophic activity) dead biomass → CO2 + H2O + energy ❑ Biogeochemical cycling of elements (C, O, N, P, S, Fe)  Microorganisms in Nature ❑Live in habitats suited to higher organisms, also in “extreme” environments ❑ extremes in temperature, pH, pressure, salinity; anoxic habitats ❑ inanimate (soil, sediment, water, food) & animate habitats (on/in animals, plants, insects) ❑ necessities for growth include available resources, suitable physiochemical conditions  Psychrophiles, thermophiles, hyperthermophiles: “extremophiles” that live in habitats of extreme temperature, including cold (e.g., deep sea, Antarctica, the Arctic), or hot habitats (e.g., compost piles, deep sea hydrothermal vents) Seawater evaporating ponds near San Francisco Bay, used to harvest “solar” salt. The red colour is due to pigments of the extreme halophile Halobacterium, an Archaeal species that inhabits the ponds. (Fig. 13.2(b), p. 423, Madigan & Martinko)  Microorganisms in nature ❑ Niche: the functional role of an organism within an ecosystem; combined description of the physical habitat, functional role, and interactions of the microorganism occurring at a given location ❑ Microenvironment: where a microorganism lives, metabolizes within its habitat ❑ physicochemical gradients ❑ spatial, temporal variability Figure illustrates O2 contours within a soil particle, measured by microelectrode. Each zone could be considered a different microenvironment ❑ Microcolonies in soil particles ❑ Very few microbes are free; most reside in microcolonies attached to soil particles ❑ Soil aggregates can contain many different microenvironments supporting the growth of several types of microbes  Environments and Microenvironments ❑Growth of microbes depends on resources and growth conditions ❑Differences in the type and quantity of resources and the physiochemical conditions of a habitat define the niche for each microbe ❑realized niche or prime niche For each organism, there exists at least one niche in which that organism is most successful ❑fundamental niche Full range of environmental conditions under which an organism can exist Resources and Conditions That Govern Microbial Growth in Nature Resources Carbon (organic, CO2) Nitrogen (organic, inorganic) Other macronutrients (S, P, K, Mg) Micronutrients (Fe, Mn, Co, Cu, Zn, Mn, Ni) O2 and other electron acceptors (NO3−, SO42−, Fe3+) Inorganic electron donors (H2, H2S, Fe2+, NH4+, NO2−) Conditions Temperature: cold→warm→hot Water potential: dry→moist→wet pH: 0→7→14 O2: oxic→microoxic→anoxic Light: bright light→dim light→dark Osmotic conditions: freshwater→marine→hypersaline  Nutrient levels and growth rates ❑ Microbial life in nature does not necessarily resemble microbial life in lab culture ❑ Entry of nutrients into an ecosystem is often intermittent ❑ Feast-or-famine existence ❑ Adaptations ❑ Accumulate reserves in times of plenty ❑ High growth rate when growth possible; quiescence when growth is not possible ❑ Periods of extended exponential growth rare in nature ❑ Distribution of resources in nature is often non-uniform ❑ Competition for resources is likely  Nutrient levels and growth rates ❑The types of microbial activities occurring in an ecosystem are a function of the number, variety, and physiological state of species present, ❑The prevailing growth conditions determine the rates of microbial activities  Microenvironments ❑Microenvironment The immediate environmental surroundings of a microbial cell or group of cells Soil particles contain many microenvironments ❑Physiochemical conditions in a microenvironment are subject to rapid change, both spatially and temporally ❑Resources in natural environments are highly variable, and many microbes in nature face a feast-or-famine existence ❑Growth rates of microbes in nature are usually well below maximum growth rates defined in the laboratory  Environments and Microenvironments ❑Competition and cooperation occur between microbes in natural systems ❑Syntrophy microbes work together to carry out transformations that neither can accomplish alone microbial partnerships are particularly important for anoxic carbon cycling metabolic cooperation can also be seen in the activities of organisms that carry out complementary metabolisms  General Ecological Concepts: Symbiotic Relationship ❑Many microbes establish relationships with other organisms (symbioses) ❑ Parasitism ❑ One member in the relationship is harmed, and the other benefits ❑ Mutualism ❑ Both species benefit ❑ Commensalism ❑ One species benefits, and the other is neither harmed nor helped  Examples of interactions between microbial populations (i) Negative effect for (one or both) interacting populations: * Competition – outcome depends on innate capabilities of nutrient uptake, metabolic rates “competitive exclusion” is one possible outcome * Antagonism – specific inhibitor or metabolic product may impede growth/metabolism of others antibiotic or bacteriocin release, lactic acid production (ii) Positive effect for (one or both) interacting populations: * Cooperative interactions - interacting microbes must share same/nearby microenvironment Syntrophy – microorganisms together carry out transformation neither can conduct alone (iii) Complementary metabolic interactions e.g., in nitrification: NH3 → NO2- (nitrosifying bacteria); NO2- → NO3- (nitrifiers) e.g., in S cycling: anaerobic sulfate reducing bacteria (SO42- → H2S) provide substrate for microaerophilic sulfide-oxidizing bacteria (H2S → S0)  Surfaces and Biofilms ❑Surfaces are important microbial habitats Typically offering microbes greater access to nutrients and protection from predation and physicochemical disturbances Nutrients adsorb to surfaces Attachment to a surface also offers cells a means to remain in a favorable habitat, modify the habitat by their own activities, and not be washed away  Surfaces and Biofilms Biofilms ❑ assemblages of bacterial cells adhered to a surface and enclosed in an adhesive matrix excreted by the cells ❑ The matrix is typically a mixture of polysaccharides. ❑ Biofilms trap nutrients for microbial growth and help prevent detachment of cells in flowing systems.  Surfaces and Biofilms ❑ Biofilm formation is initiated by attachment of a cell to a surface followed by expression of biofilm-specific genes. ❑ Genes encode proteins that synthesize intercellular signaling molecules and initiate matrix formation.  Surfaces and Biofilms Pseudomonas aeruginosa ❑ Biofilm producer ❑ Intracellular communication (quorum sensing) is critical in the development and maintenance of a biofilm ❑ The major intracellular signaling molecules are acylated homoserine lactones (AHL) ❑ Both intraspecies signaling and interspecies signaling likely occur in biofilms  Surfaces and Biofilms Biofilm: a community of microorganisms embedded in an organic polymer matrix (extracellular polymeric substances, EPS), adhering to a surface ❑ Surfaces offer microbes greater access to nutrients and protection from predation and physicochemical disturbances ❑ Nutrients adsorb to surfaces and microbial cells can attach to surfaces ❑ Attachment to a surface offers cells a means to remain in a favorable habitat, Time modify the habitat by their own activities, and not be washed away ❑ Physicochemical gradients within mature biofilm result in a number of potential microenvironments within a small area Surfaces and Biofilms  Biofilms: Heterogeneity Mature biofilm is a complex, dynamic community of microorganisms. Heterogeneity: differences in metabolic activity and locations of microbes. Interactions occur among the attached organisms. E.g. Exchanges take place metabolically, DNA uptake and communication.  Biofilms: Common in Nature Most microbes grow attached to surfaces (sessile) rather than free floating (planktonic) Biofilm: attached microbes are members of complex, slime enclosed communities. Biofilms are ubiquitous in nature in water. Can be formed on any conditioned surface. Bacterial microcolonies developing on Natural biofilm on a leaf surface a microscope slide immersed in a river ❑ cell colour indicates depth in biofilm: red (surface) (phase contrast microscopy) → blue (18 μm deep) (confocal laser scanning microscopy) Biofilm developed on a stainless-steel pipe ❑ stained with DAPI (fluorescent; interacts with nucleic acids) ❑ note water channels through biofilm  Evidence of a dental biofilm ❑left front tooth exposed to sucrose solution for 5 min while right served as a control ❑both then stained with iodine solution ❑brown colouration results from reaction of iodine with extracellular glucans (EPS) produced by the sucrose- supplied biofilm  Microbial Mats ❑ Microbial mats can be considered extremely thick biofilms, and they usually consist of a complex community of different microorganisms. ❑ Built by phototrophic and/or chemolithotrophic bacteria ❑ For example, cyanobacterial mats consist of primary producers (the cyanobacteria) at the surface and an assortment of heterotrophic consumers below ❑ Microbial mats are also found in metal-rich, low-pH environments such as those found associated with mining and some geothermal vent systems where iron-oxidizing Bacteria and Archaea may cause environmental damage when the acidity and metals released by their metabolisms pollute other aquatic ecosystems ❑ Impacted by the Diel cycle Microbial mat core from an alkaline hot spring (Yellowstone National Park)  Iron-Oxidizer Microbial Mats ❑ Chemolithotrophic mats are also common ❑ Most of these consist of sulfur-oxidizing bacteria that reside at the O2/hydrogen mat of iron-oxidizing bacteria attached to rocks in the Rio Tinto, Spain sulfide (H2S) interface on marine sediment surfaces. ❑ Such organisms are important primary producers that carry out carbon fixation in permanently dark ecosystems where photosynthesis is not possible. As ferrous (Fe2+)-rich water from metal mining activities flows over and through the biofilm, the organisms oxidize Fe2+ to Fe3+ Phototrophic Biofilms in Rivers and Streams (a)Phototrophic biofilms colonizing the rocky bottom of a stream flowing from the Rhone Glaciers, Switzerland (b)Cyanobacteria attached to the river rocky in “tower-like” clusters with apical oxygen bubbles  Thioploca Mats (a, c) Filaments of the large sulfur-oxidizing chemolithotroph Thioploca in the Bay of Concepción off the Chilean coast. (b) Thioploca form bundles of 10 to 20 filaments (trichomes) held together by a gelatinous sheath *Two species of Thioploca commonly inhabit the same bundle: T. chileae and T. araucae  Biofilms: Advantages to Microbes ❑ Bacteria form biofilms for several reasons: ❑ attachment to surface, even in flow systems ❑ nutrient trapping ❑ Self-defense ❑ Biofilms resist physical forces that sweep away unattached cells, phagocytosis by immune system cells, and penetration of toxins (e.g., antibiotics), predators ❑ Allows cells to remain in a favorable niche (cooperative interactions possible) ❑ Allows bacterial cells to live in close association with one another Biofilms: Disadvantages to Microbes ❑highly competitive ❑localized biomass can be efficiently preyed upon, infected by viruses ❑Reduces motility ❑Can you think of any other?  Biofilms: Advantages to Humans ❑Exploitation of biofilms: slow sand filtration (water purification) microbial leaching of low-grade ores; Fermentation, vinegar production etc. Can you think of any other?  Biofilms: Disadvantages to Humans ❑Biofilms have been implicated in several medical and dental conditions. periodontal disease, kidney stones, tuberculosis, Legionnaires’ disease, Staphylococcus infections, cystic fibrosis due to P. aeruginosa, implanted medical devices ❑In industrial settings, biofilms can slow the flow of liquids through pipelines, high microbial numbers in potable water distribution systems; accelerated corrosion of pipelines and structural steelwork; increased drag on ship’s hull ❑Surface colonization and biodegradation of plastic alters the surface chemistry, density, and sinking rates of MPs (Microplastics) ❑Antibiofilm agents are available (https://www.future- science.com/doi/pdfplus/10.4155/fmc.15.7#:~:text=These%20new%20antibiofilm%20agents%2C%2 0which,human%20medicine%20in%20the%20future.)  Current Trends in Microbial Ecology ❑ Space exploration – microbes in extreme environments (hot springs, thermal vents, lithosphere) ❑ Molecular techniques – diversity of microorganisms (Carl Woese), new methods to assess presence or abundance of individual species in situ ❑ Realization that with pure culture/enrichment techniques, we know somewhere between 1-10% of existing microbial species – lots to learn! ❑ Biology of climate change, global biogeochemistry  Recent Discovery ❑ In 2008, Prof. Gary Strobel ,Montana State University and students explored the Patagonia rainforest they found an endophytic fungus inside the tissues of the Ulmo tree. The fungus Gliocladium roseum liberates a number of volatile compounds in the air, the mixture is similar to diesel fuel and can be produced when grown in the lab with good yields on cellulose - dubbed mycodiesel ❑ Also, Prof. Scott Strobel and a group of Yale students in 2012 found in the Amazon rainforest a fungus Pestialotiopsis microspora that degrades polyurethane (plastic). The fungus is able to survive on polyurethane alone under anaerobic conditions  History of Microbial Ecology ❑The term “microbial ecology” really wasn’t in common use until the late 1960s ❑Why? ❑Microbial ecology has its roots in microbiology, rather than ecology ❑The history of the field is largely a transition from laboratory pure cultures to studying organisms in nature…  Louis Pasteur (1822-1895) ❑ “basic” vs. “applied” science ❑ fermentation = biological process carried out by microorganisms. ❑ Germ theory = foundation of brewing of beer, wine-making, and pasteurization. ❑ Nature of contagious diseases: potato blight, silkworm diseases, and anthrax. ❑ Immunization (anthrax, rabies) ❑ Public experiments! "Imagination should give wings to our thoughts but we always need decisive experimental proof, and when the moment comes to draw conclusions and to interpret the gathered observations, imagination must be checked and documented by the factual results of the experiment."  Robert Koch (1843-1910) ❑ Discovered the Bacillus strains that cause cholera and anthrax ❑ Agar media for pure cultures (earlier had tried sliced boiled potatoes!) ❑ Pure culture paradigm: isolate an ❑ Disease and medical organism and see what it microbiology does ❑ Pure culture paradigm  Pure Culture Paradigm ❑ Extremely important conceptual development in microbiology (and in microbial ecology, too) ❑ Remove organisms from complex communities ❑ Isolate key processes ❑ Obtain reproducible results ❑ This method is still used today ❑ Attitude of Koch’s time: ❑ “Work with impure cultures yields nothing but nonsense and Penicillium glaucum“ (Oscar Brefield 1881) Sir Alexander Fleming (1929), examining exactly such an impure culture (Staphylococcus culture contaminated by Penicillium), led to the discovery of penicillin. Agar petri dish zone of no bacterial growth, Staphylococcus due to penicillin colonies produced by fungus Penicillium contaminant Interference competition! classic ecological process  Sergei Winogradsky (1856-1953) ❑ Isolated nitrifying bacteria ❑ Winogradsky column: microbial communities develop along a gradient of oxygen tension; method still used today ❑ Described oxidation of hydrogen sulfide, sulfur, ferrous iron ❑ …all leading to the concept of chemoautotrophy – deriving ❑ Bacteria: central in element energy from chemical oxidation of inorganic compounds and transformations carbon from CO2 ❑ Founder of soil microbiology  Martinus Beijerinck (1851-1931) “The way I approach microbiology...can be concisely stated as the study of microbial ecology, i.e., of the relation between environmental conditions and the special forms of life corresponding to them” Founder of the Dutch Delft School of Microbiology  Martinus Beijerinck (1851-1931) ❑ “a man of science does not marry” ❑ Isolated N fixers and S reducers ❑ ‘Microbial ubiquity’: all microorganisms are everywhere; conditions and resources determine who flourishes ❑ Enrichment culture: growth medium tailored to suit particular metabolic function ❑ With Winogradsky, recognized that microbes are the major players in element transformations ❑ Led to field of global biogeochemistry Albert Jan Kluyver (1888-1956) ❑ Student of Beijerinck ❑ Microbial physiology ❑ Comparative approach ❑ Unifying metabolic features among microbes ❑ Leader of the Dutch school after Beijerinck  Cornelius Bernardus van Niel (1897-1985) ❑ Student of Kluyver, third in the Dutch Delft School ❑ Isolated purple sulfur bacteria ❑ Major contribution, chemistry of photosynthesis: ❑ H2A + CO2 → CH2O + 2A + H2O where A can be S or O ❑ Extended model green plants; oxygen from water, not from CO2 ❑ Also, chemistry of denitrification, definition of prokaryote in 1961 (with R. Stanier) ❑ Taught lab course focusing on studying microbes from nature (first course in microbial ecology?) ❑ Philosophy of hypothesis testing, falsification “moving from clearly erroneous to more ‘correct’, but never immutable conclusions”  Robert E. Hungate (1908-2004) ❑ Student of van Niel ❑ Developed methods for isolating anaerobes ❑ Devised culture methods that select using natural substrates, rather than guesses about what organisms eat ❑ Microbiology of guts of rumen, termites ❑ ASM president when ❑ AKA “Grampa Bob” “Environmental Microbiology” and “Microbial Ecology” formally recognized  Other contributions ❑1960s: Ronald Atlas, Richard Bartha ❑ Studies of petroleum degradation ❑ Led to new field of bioremediation, ❑ Extended to many other pollutants: DDT, PCBs, mercury, selenium, industrial solvents ❑1970s fuel-shortage: ❑ Shortage in N fertilizer ❑ Sparked interest in the biology of nitrogen fixers  Methods for studying microbial ecology [Part 2] Dr. Stacy A-M Stephenson-Clarke [email protected] MICR3213 [BC31M]: Applied and Environmental Microbiology  QUOTE OF THE DAY 2 9/11/2024 Add a footer  Learning Objectives – Part 2 ❑ Explain how microbial ecologists measure community activity ❑ Describe why microelectrode measurements are important tools in the study of microbial community activity ❑ Compare the application of traditional stable isotope analysis with stable isotope probing ❑ Assess the advantages and disadvantages of measuring in situ mRNA abundance ❑ List the type of data that can be generated by MAR-FISH and compare this with functional gene arrays ❑ Predict which techniques would be appropriate for assessing the quantity versus diversity versus activity of a microbial community 3 9/11/2024 Add a footer  Outline I. Experimental design and sampling II. Staining techniques: Culture-Independent Microscopic Analyses of Microbial Communities III. DNA-based techniques: Culture-Independent Genetic Analyses of Microbial Communities IV. Measuring Microbial Activities V. Linking Genes and Functions to Microorganisms IV. Culture Independent Methods: Measuring Microbial Activities in Nature A.Chemical Assays, Radioisotopic Methods, and Microsensors B. Stable Isotopes C.Linking Genes and Functions to Specific Organisms: Stable Isotope Probing and Single-Cell Genomics D.Linking Genes and Functions to Specific Organisms: SIMS, Flow Cytometry, and MAR-FISH  A. Measuring Microbial Activities in Nature: Chemical Assays, Radioisotopes, and Microsensors In many studies, direct chemical measurements are sufficient Higher sensitivity can be achieved with radioisotopes Proper killed cell controls must be used A. Measuring Microbial Activities in Nature: Chemical Assays Vast majority of microorganisms in nature have not been cultured Termed viable but not culturable (VBNC) Primary source of information for these microorganisms is their biomolecules; Lipids, proteins and DNA/RNA  A. Measuring Microbial Activities in Nature: Chemical Assays Phospholipid fatty acids (PLFA) ❑ Lipids Extract, concentrate, structural analysis ❑ Quantitative ❑ Insight into viable biomass, community composition, nutritional-physiological status, evidence for metabolic activity  A. Measuring Microbial Activities in Nature: Chemical Assays - PLFA Designated based on: The total number of C atoms Degree of unsaturation (double bonds) Position of the double bonds Branching patterns Examples: 16:0 = 16 carbons, no double bond 18:25 = 18 carbons, 2 double bonds at the 5th position from the aliphatic end i15:0 = 15 carbons, no double bond with iso-branching a15:0 = 15 carbons, no double bond with ante iso-branching  A. Measuring Microbial Activities in Nature: Chemical Assays - PLFA Some ecologically important patterns have been recognized: Ratios of i15:0 and a15:0 PLFA to 16:0 PLFA is a useful index of the proportion of bacteria and eukarya in the community, [iso – methyl group on second-to-last C; ante iso- methyl group on third-to-last C] Also, ratios of trans- and cis- isomers of saturated to unsaturated fatty acids may indicate physiological conditions of organisms or environmental stress. Proportion of diglycerides – a function of cell death/lysis/action of phospholipases  A. Measuring Microbial Activities in Nature: Chemical Assays, Radioisotopic Methods, Microsensors, and Nanosensors In many studies, direct chemical measurements are sufficient Lactate, SO42−, and H2S can all be measured with high sensitivity by chemical assay Higher sensitivity can be achieved with radioisotopes Proper killed cell controls must be used to separate the chemical action of microbes from that of abiotic processes In some activity measurements, it is useful to inhibit or encourage the activities of certain organisms Acetylene as an inhibitor or alternative substrate A. Measuring Microbial Activities in Nature: Microbial Activity Measurements A. Measuring Microbial Activities in Nature: The Acetylene Reduction Assay of Nitrogenase Activity in Nitrogen-Fixing Bacteria  A. Measuring Microbial Activities in Nature: Microsensors A microelectrode is a conductor through which electric current is passed, between a metallic part and a nonmetallic part of an electrical circuit Small glass electrodes with the tip sizes ranging from 2 to 100 μm in diameter Electrochemical reactions due to the presence of a substrate change the current – a substance is detected Can measure pH, oxygen, N2O, CO2, H2,H2S, and others  A. Measuring Microbial Activities in Nature: Microsensors Microsensors/Microelectrode Small glass electrodes, quite fragile Can measure a wide range of activity pH, oxygen, CO2, and others can be measured Electrodes are carefully inserted into the habitat (e.g., microbial mats) Measurements taken every 50–100 mm Use of Microelectrodes: Microbial Mats Babauta et al.(2014). Front. Microbiol. Microelectrodes: Oxygen Nanosensor Analysis of Coral Photosynthetic Activity ▪ Calibration of nanosensor response to different dissolved oxygen concentrations using a fragment of a coral skeleton painted with the nanosensor octaethylporphine ketone platinum (II). ▪ (b) Living coral response to transition from darkness (top panel) to light (bottom panel). ▪ Note how the oxygen-depleted region due to coral respiration in the dark panel (arrow) quickly becomes oxygenated in the light. A. Measuring Microbial Activities in Nature: Stable Isotopes and Stable Isotope Probing ❑Stable isotopes: nonradioactive isotopes of an element ❑used to study microbial transformations in nature 1H 2H 12C 13C 14N 15N 32S 33S 34S 36S B. Measuring Microbial Activities in Nature: Stable Isotopes Element Isotopes Abundance Hydrogen 1H, 2H 1H = 99.985% 2H = 0.015% Carbon 12C, 13C 12C = 98.89% 13C = 1.11% Nitrogen 14N, 15N 14N = 99.633% 15N = 0.366% Oxygen 16O, 17O, 18O 16O = 99.759% 17O = 0.037% 18O = 0.204% Sulfur 32S, 33S, 34S, 36S 32S = 95.00% 33S = 0.76% 34S = 4.22% 36S = 0.014% B. Measuring Microbial Activities in Nature: Stable Isotopes ❑ Stable isotope ratios (e.g.,13C/12C) are measured using a mass spectrometer. Three masses of CO2 (44/45/46) are measured to determine the amount of 13C and 12C in a sample 12C+16O+16O = 44 13C+16O+16O = 45 12C+18O+16O = 46 B. Measuring Microbial Activities in Nature: Stable Isotopes Not radioactive but metabolized differentially by microorganisms Enzymes typically favour the lighter isotope Two methods: stable isotope fractionation (SIF) and stable isotope probing (SIP)  B. Measuring Microbial Activities in Nature: Stable Isotope Fractionation Isotope fractionation Carbon and sulfur are commonly used Lighter isotope is incorporated preferentially over heavy isotope Indicative of biotic processes Isotopic composition reveals its past biology (e.g., carbon in plants and petroleum) The activity of sulfate-reducing bacteria is easy to recognize from their fractionation of sulfur in sulfides Enzyme substrates Fixed carbon Enzyme that fixes CO2 12CO 2 12C organic 13CO 13C 2 organic  Stable Isotope Probing VIDEOS: 1. https://www.jove.com/v/2027/dna-stable-isotope- probing- dnasip?utm_source=youtube&utm_medium=social_global &utm_campaign=reseach-videos-2022 2. https://www.youtube.com/watch?v=fe3wyesvRAw  B. Linking Genes and Functions to Specific Organisms: Stable Isotope Probing Stable isotope probing (SIP): links specific metabolic activity to diversity using a stable isotope Microorganisms metabolizing stable isotope (e.g., 13C) substrate will incorporate it into their DNA DNA with 13C can then be used to identify the organisms that metabolized the 13C SIP of RNA also possible B. Linking Genes and Functions to Specific Organisms: Stable Isotope Probing B. Linking Genes and Functions to Specific Organisms: Stable Isotope Probing Principles: ▪ Incorporation of 13C-labeled substrate into cellular biomarkers (e.g. nucleic acids); ▪ Separation of labelled from unlabeled nucleic acids by density gradient centrifugation; ▪ Molecular identification of active populations carrying labelled nucleic acid Advantage: Allows the identification of active microorganisms without the use of radioactive isotopes. B. Linking Genes and Functions to Specific Organisms: Stable Isotope Probing Disadvantages: ▪ Possible biases caused by the incubation with the isotope ▪ Cycling of the stable isotope within the microbial community.  C. Linking Genes and Functions to Specific Organisms: SIMS Analysis of cells by secondary ion mass spectrophotometry (SIMS) Detection of ions released from sample placed under focused high energy primary ion beam High-energy ion beam impacts a sample Secondary ions are released (sputtering) Mass spectrometry of secondary ions Data generated from mass spectrometer reveals elemental and isotopic composition of released materials (secondary ion) FISH-SIMS – traces incorporation of different elements or isotopes into individual cells of specific cell populations (previously labelled with FisH probes)  C. Linking Genes and Functions to Specific Organisms: SIMS NanoSIMS SIMS devices that yield information on single cells Uses multiple detectors to provide simultaneous analysis of ions Allows for a two-dimensional image of the distribution of specific ions on the sample surface reveals info on single cells using O2 beam (generates positive secondary ions to analyse metals (e.g., Fe, Mg) and Cs+ beam (generates negative secondary ions for analysis of cellular elements e.g., C, N, P, S, O, H and halogens)  Bi = Bismuth Cs = Caesium ChiuHuang et al. (2014). ASME. J. Nanotechnol. Eng. Med. 5(2):021002-021002-5 Fluorescence and NanoSIMS images of a microbial consortium consisting of filamentous cyanobacteria (Anabaena sp. strain SSM-00) and alphaproteobacteria (Rhizobium sp. strain WH2K) attached to heterocysts Behrens et al.( 2008) Appl. Environ. Microbiol. 74:10 3143-3150 37 9/11/2024 Add a footer C. Linking Genes and Functions to Specific Organisms: Linking Functions to Specific Organisms Raman Microspectroscopy Be used to characterize the molecular and isotopic composition of single cells by nondestructive illumination with monochromatic light generated by a laser When combined with confocal microscopy, Raman spectrometers can determine the elemental composition and appearance of a single microbial cell *Confocal microscopy - increases optical resolution and contrast by use of a spatial pinhole to block out-of-focus light when forming an image  C. Linking Genes and Cellular properties to Individual Cells: Flow Cytometry Flow cytometry and multiparametric analysis Natural communities contain large populations Flow cytometer examines specific cell parameters very fast as they pass through a detector Cell size Cell shape Fluorescence Parameters can be combined and analyzed (multiparametric analysis) Example: resolved two populations of marine cyanobacterium (Prochlorococcus and Synechococcus) based on differences in size and fluorescence in the late 1980s Video: https://www.youtube.com/watch?v=mcnFTjcmykE C. Linking Genes and Functions to Individual Cells: MAR-FisH Radioisotopes are used as measures of microbial activity in a microscopic technique called microautoradiography (MAR) Radioisotopes can also be used with FISH microautoradiography FISH (MAR-FISH) combines phylogeny with activity of cells Simultaneous assessment of metabolic activity and phylogenetic identity of microbes of interest at the level of a single cell in complex microbial communities. C. Linking Genes and Functions to Specific Organisms: MAR-FISH Assesses both activity and identity Identifies organisms metabolizing radiolabelled substance Other techniques plus FISH- ISRT-FISH (in situ reverse transcriptase PCR/FISH) used to examine gene expression CARD-FisH Cottrell, M. (2016). Website accessed at https://www.ceoe.udel.edu/our- people/profiles/mattcott C. Linking Genes and Functions to Specific Organisms: FISH-MAR: Methodology ❑ When left in the dark radioactive decay of incorporated substance exposes silver grains in the emulsion. ❑ Appear as black dots within and around the cells. C. Linking Genes and Functions to Individual Cells: MAR-FisH ©Cleber Ouverney  C. Linking Genes and Functions to Specific Organisms: MAR-FISH Radioisotopes are used as measures of microbial activity in a microscopic technique called microautoradiography (MAR) Radioisotopes can also be used with FisH Microautoradiography FISH (MAR-FISH) Combines phylogeny with activity of cells 45 9/11/2024 Add a footer Methods for Studying Microbial Ecology [Part 1] MICR3213 [BC31M]: Applied and Environmental Microbiology Dr. Stacy Stephenson-Clarke [email protected]  Learning Objectives – Part 1 ❑ Explain why, to the best of our knowledge, most microorganisms resist culturing in the laboratory ❑ Describe the use of enrichment cultures ❑ Explain why microbial communities are often investigated in situ ❑ Describe why, when, and how FISH and CARD-FISH are used ❑ Summarize how PCR is used to take a microbial census ❑ Explain why and how DGGE is used ❑ Describe the use of phylochips to assess microbial communities ❑ Differentiate metagenomics from metaproteomics 2 9/11/2024  Learning Objectives – Part 2 ❑ Explain how microbial ecologists measure community activity ❑ Describe why microelectrode measurements are important tools in the study of microbial community activity ❑ Compare the application of traditional stable isotope analysis with stable isotope probing ❑ Assess the advantages and disadvantages of measuring in situ mRNA abundance ❑ List the type of data that can be generated by MAR-FISH and compare this with functional gene arrays ❑ Predict which techniques would be appropriate for assessing the quantity versus diversity versus activity of a microbial community 3 9/11/2024 Add a footer  Methods in Microbial Ecology ❑ The major components of microbial ecology are biodiversity and microbial activity. ❑ To study biodiversity, microbial ecologists must identify and quantify microorganisms in their habitats. ❑ Knowing how to do this is often helpful for isolating organisms of interest as well, which is another goal of microbial ecology. ❑ To study microbial activity, microbial ecologists must measure the metabolic processes that microorganisms carry out in their habitats. 4 9/11/2024 Add a footer  ❑ Virtually all microorganisms exist as parts of complex communities that interact through metabolic cooperation. ❑ Since complex metabolic interactions are not easily resolved, the microscope is an essential tool for first identifying possible cooperation based on the colocalization of different species. ❑ However, even the simplest of microbial communities is composed of tens if not hundreds of different species. ❑ How it is possible to visualize the distribution of individual species in such a mixture? 5 9/11/2024 Add a footer  Microbial Ecology Culturing Methods Microbial ecology is an interesting and different take on the standard in-lab culturing methods. The idea takes into account the many microbes that cannot be cultured—so how can we change what we’re doing to study them when we can’t grow them? This section introduces a number of tools/methods for studying microbes in their natural environments (in situ) and when they can’t grow in the lab setting.  CLASI-FISH (combinatorial labeling and spectral imaging–FISH) ❑ can image more than 100 different species simultaneously. ❑ hybridizes each cell with a combination of probes specific for that species but labeled with different fluorescent dyes, giving each cell a unique fluorescent spectral signature. ❑ Since the resulting fluorescence at each wavelength is a linear combination of emissions from each fluorescent dye, statistical analysis can determine what combination of dyes produced the emission spectrum Scraping of the human tongue and therefore identify the contributing species. ❑ Brown in the photo is human tissue; the bacteria are: red, Actinomyces spp.; green, Streptococcus spp.; blue, Rothia spp.; yellow, Neisseria spp.; and magenta, Veillonella spp. 7 9/11/2024 Add a footer  Methods in Microbial Ecology Understanding the biodiversity of microbes, and interactions in communities Measurement of microbial activities, and monitoring of effects on ecosystems There are many challenges associated with studying microbial systems  Methods in Microbial Ecology Interdependent series of inquiries involving traditional, laboratory-based analyses, metagenomics, and in situ biogeochemical assessments. Techniques to culture microbes (gold standard). Methods used to measure the activities in nature.  Methods in Microbial Ecology Culture-dependent methods Enrichment and isolation Culture methods Enrichment bias MPN Culture-independent methods Most accurately represent populations and communities Our focus  Culture-dependent methods: Enrichment Enrichment cultures Can prove the presence of an organism in a habitat Cannot prove that an organism does not inhabit an environment The ability to isolate an organism from an environment says nothing about its ecological significance Enrichment bias Microorganisms cultured in the lab are frequently only minor components of the microbial ecosystem Reason: the nutrients available in the lab culture are typically much higher than in nature Dilution of inoculum is performed to eliminate rapidly growing, but quantitatively insignificant, “weed” species  Enrichment Culture Microbiology Enrichment Culture Outcomes Successful enrichment cultures have appropriate resources (nutrients) and conditions (temperature, pH, oxygen, osmotic considerations) that are needed for the target organisms to grow Enrichment cultures can demonstrate the presence of an organism in a habitat They cannot prove that an organism does not inhabit an environment Note: The ability to isolate an organism from an environment says nothing about its ecological importance or relative abundance in nature Some Enrichment Culture Methods for Phototrophic Bacteria (Main C Source, CO2) Incubation in air Incubation condition Organisms enriched Inoculum N2 as nitrogen source Cyanobacteria Pond or lake water; sulfide-rich muds; stagnant water; raw sewage; moist, decomposing leaf litter; moist soil exposed to light N O3− as nitrogen source, 55°Celsius Thermophilic cyanobacteria Hot spring microbial mat Anoxic incubation Incubation condition Organisms enriched Inoculum H2 or organic acids; N2 as sole Purple nonsulfur bacteria, Same as above plus hypolimnetic lake water; pasteurized soil nitrogen source heliobacteria (heliobacteria); microbial mats for thermophilic species H2S as electron donor Purple and green sulfur bacteria Blank Fe2+, NO2− as electron donor Purple bacteria Blank  Reasons Microbes May Be “Unculturable” Potential Problem Example Method Designed to Overcome Microbe is slow growing. Incubation times of weeks to months Microbe is present in very low abundance. Extinction cultures, using many replicates Different microbes in same habitat are Remove other microbes from the sample by physical methods physiologically very similar. such as filtration or density-gradient centrifugation or use OR extinction cultures. Inhibition by other microbes in a mixed culture Fastidious growth requirement Assess growth requirements of similar microbes, if known. Use annotation of metagenomic sequences to infer nutritional capabilities and requirements; grow in diffusion chambers that allow influx of small molecules from natural environmental samples without microbial contamination. Cross-feeding or communication signals from Co-cultivate strains; use diffusion chambers; grow in other microbes are needed. conditioned spent medium of “helper” microbe. Triggers for growth or exit from a dormant state Add known growth triggers such as N-acetylmuramic acid. are not present.  Three Reasons for Culture Discrepancy ❑ Viable but nonculturable (VBNC) Motility, dividing cells, grows in nature, or stains with dyes for living cells. ❑ Great plate count anomaly (GPCA) Discrepancy between number of microbial cells observed by microscopic examination and number of colonies that can be cultivated from the same natural sample. ❑ Not the right conditions for growth of environmental microbes. Enrichment cultures. ©Dr. Rita B. Moyes  Classical Procedures for Isolating Microbes Pure cultures contain a single kind of microorganism Can then be used for molecular and physiological experiments Streak plate A well-isolated colony is selected and restreaked several successive times to obtain a pure culture  Classical Procedures for Isolating Microbes ❑ Agar dilution tubes are mixed cultures diluted in molten agar ❑useful for purifying anaerobic organisms; tubes sealed with paraffin + mineral oil ❑Most-probable-number technique ❑serial 10x dilutions of inoculum in a liquid medium ❑used to estimate number of microorganisms in food, wastewater, and other samples  Most Probable Number (MPN) Technique for estimating the number of microbes in a natural sample. ❑Samples include food or water. ❑Serial dilutions and observation of growth. ❑Microbe must be capable of growth in laboratory. Culture-dependent methods: Approaches to Microbial Growth Use of unusual electron donors and acceptors. Prolonged time in culture. Unusual cell densities in nature (too low to appear turbid). Allowing bacteria to exchange secreted metabolites and signaling molecules through filters or gels. Extinction culture technique. Dilution of natural sample to fewer than 10 cells. Incubation and screening for growth.  Culture-dependent methods: Selective Single-Cell Isolation Laser Tweezers, Flow Cytometry, Microfluidics, and High-Throughput Methods Problem: enrichment bias from traditional methods limits understanding of microbes in nature New techniques have been developed to address this problem when isolating microbes from nature Premise: Microbes have both a realized niche and a fundamental niche The fundamental niche indicates where an organism could live, while the realized niche is where an organism does live, despite resource limitations and competition Culture-dependent methods: Microbial Growth—Flow Cytometry Technique for counting and examining a mixture of cells by suspending them in a stream of fluid and passing through an electronic detector Procedure: Cells are tagged with a fluorescent dye and injected into a flowing stream of fluid. As the diameter of the stream narrows, one cell at a time is forced through a thin tube, detected by a laser beam. Can detect and sort cell population based on cell size, shape, and morphology. Culture-dependent methods: Isolation of Individual Cells ❑ Optical/Lazer tweezers isolating slow-growing bacteria from mixed cultures; physically separating individual cells for culture Laser beam used to drag microbe away from its neighbors if the sample is not an axenic (pure) culture. Can be coupled with staining techniques for identification of cells ❑ Isolation can be followed by analysis of organism’s DNA for phylogenetic analysis or single cell genomic sequencing. Methodological Pipeline for High-Throughput Cultivation of Previously Uncultured Microorganisms Microfluidic Platform for Cultivation ❑ carries the high-throughput concept even further by using microfabrication technology to combine channels and wells for fluid transfer and collection on a miniaturized platform. ❑ One such device is less than 10 centimeters long yet holds 3200 nanoliter-sized wells, with each well serving as a small culture vessel  Methods: Identity vs Function Culture-Independent Microscopic Analyses: Viability staining, Fluorescent Protein Tags (e.g. GFP), FISH, CARD-FISH Culture-Independent Genetic Analyses of Microbial Communities: PCR, DGGE, T- RFLP, ARISA, Microarrays, Metagenomics, Metatranscriptomics and Metaproteomics Measuring Microbial Activities: Chemical assays, Radioisotopic methods (e.g. 14C, 35S ), Microsensors (e.g. microelectrodes), Stable isotopes (e.g. SIP, SIF) Linking Genes and Functions/Activity to Microorganisms: SIMS, NanoSIMS, Flow Cytometry, MAR – FISH, DNA-SIP  Outline I. Staining techniques: Culture-Independent Microscopic Analyses of Microbial Communities II. DNA-based techniques: Culture-Independent Genetic Analyses of Microbial Communities III. Measuring Microbial Activities IV. Linking Genes and Functions to Microorganisms ❖ SEE BROCK Biology of Microorganisms 16th Ed – Chapter 19 ❖ https://www.researchgate.net/publication/51905295_Culture- independent_methods_for_studying_environmental_microorganisms_Methods_appli cation_and_perspective Staining Techniques: Examination of Microbial Community Structure ❑ The most direct way to assess microbial community structure is to directly observe communities in nature. Assessment can be done in situ using immersed slides. Assessment also by electron microscope grids placed in locations of interest and then observed later.  I. Culture-Independent Microscopic Analyses: General Staining Methods Viability stains: enumerate and differentiate between live and dead cells (esp. aquatic environs) Two dyes are used Stains based on integrity of cytoplasmic membrane Green cells are live; penetrates all cells Red (contains propidium iodidez) cells are dead; penetrate i]unintact membrane Limitation: Can have issues with nonspecific background staining in environmental samples  i. Culture-Independent Microscopic Analyses: General Staining Methods Fluorescent staining using ', 6-diamidino-2-phenylindole (DAPI), acridine orange (AO), or SYBR Green I (SYBR) DAPI-stained cells fluoresce bright blue (a); 400 nm AO-stained cells fluoresce orange or greenish orange (b); 500 nm SYBR-stained cells fluoresce green (c); 497 nm Fluoresce under UV light and are used for the enumeration of microorganisms in environmental, food and clinical samples Nonspecific and stain nucleic acids such as DNA (A-T rich regions) Limitation: Cannot differentiate between live and dead cells due to non-specific staining  I. Culture-Independent Microscopic Analyses: General Staining Methods Fluorescent tags/proteins Fluorescent labelled antibodies Means of identifying or tracking microbes Fusion proteins (e.g. GFP gene) expressed in engineered cells Assess the effect of disturbances in microbial populations I. Culture-Independent Microscopic Analyses: General Staining Methods Green fluorescent protein can be genetically engineered into cells to make them autofluorescent. Can be used to track live bacteria and bacterial processes such as infections Can act as a reporter gene to identify when a particular promoter is active Note that GFP is not a true staining method but relies instead on the expression of the gfp gene  Limitation of Microscopy Inherent limitations exist for the use of microscopy as the sole research tool. Staining methods do not accurately reveal the astounding diversity of microorganisms, which may appear identical but are genetically distinct Thus, microscopic techniques are often coupled to or supplemented with molecular-based tools that help reveal phylogenetic diversity. 33 9/11/2024 Add a footer  Article: FisH Diversity 34 9/11/2024 Add a footer  I. Culture-Independent Microscopic Analyses: FisH (Fluorescence in situ Hybridisation) Due to their specificity, nucleic acid probes can be used as tools for identifying and quantifying microorganisms Fluorescent nucleic acid probe: DNA or RNA complementary to a sequence in a target gene or RNA Uses fluorescently labeled nucleotide probes to label whole cells Typically, rRNA sequence is used to design probes specific to an organism NB: The FisH method overlaps with culture-independent methods used to link genes to microbial function Organisms identified based on their phylogeny  I. Culture-Independent Microscopic Analyses :Molecular Approaches to Staining Fluorescent in situ hybridization (FISH) When hybridized, probe fluoresces; detected by epifluorescence microscopy. Application: Used in microbial ecology, food industry, and clinical diagnostics Video: https://www.youtube.com/watch?v=b81DcJ C1jAs Fluorescent In Situ Hybridization (FISH) Assay – YouTube ©Seana Davidson  I. Culture-Independent Microscopic Analyses: FisH (Fluorescence in situ Hybridisation) FISH technology can also employ multiple phylogenetic probes (colors specific to organism, species, strains etc) Use different colored dyes to label different cells different colors Photomicrographs: (a) phase contrast and (b) phylogenetic FISH, are of the same field of cells. ❑ Advantage: Can be used for All cells look similar by phase-contrast microscopy identification & quantitative analysis However, the phylogenetic stains reveal that there ❑ Can you think of any possible are two genetically distinct types (stains yellow and disadvantage(s)? blue).  I. Culture-Independent Microscopic Analyses: FisH - Methodology To check whether a particular organism exists in a sample and in what proportion: Find out the sequence of your organism (from database such as NCBI) Make a probe (oligonucleotide) that is complementary to the rRNA of the organism and label it with fluorescent probe. Fix your sample on a microscope slide and wash with labelled probe. Look down a fluorescence microscope and see in which cells the fluorescent probe has bound I. Culture-Independent Microscopic Analyses: FISH Methodology I. Culture-Independent Microscopic Analyses: Usefulness of FISH Method Can identify a particular species, strain, eco- or phylotype. Useful in clinical diagnostics and food microbiology. Variation → CARD-FISH (Catalyzed reported deposition-FISH) Modification of FISH used to amplify the signal produced by microbe cells in low numbers. Couple fluorescent probe with enzyme that makes lots of fluorescent product when exposed to substrate.  I. Culture-Independent Microscopic Analyses: CARD-FISH Catalyzed reporter deposition FISH Used to measure gene expression in organisms in a natural sample Enhances the fluorescence signal for detection Useful in phylogenetic studies of prokaryotes that may be growing slowly (e.g. microbes inhabiting open oceans of low temp. and nutrient conc.) with few ribosomes and few mRNA Nucleic acid probe contains enzyme peroxidase conjugate instead of fluorescent dye After hybridization, preparation treated with fluorescently labelled compound (tyramide), which is substrate for peroxidase Substrate is then converted to reactive intermediate that covalently binds to adjacent proteins Signal sufficiently amplified to be detected by fluorescence microscopy I. Culture-Independent Microscopic Analyses: CARD-FISH https://www.arb-silva.de/fish-probes/fish-protocols/ I. Culture-Independent Microscopic Analyses: CARD-FISH Archaeal cells in this preparation fluoresce intensely (green) relative to DAPI- stained cells (blue).  I. Culture-Independent Microscopic Analyses: BONCAT-FISH Terms Biorthogonal: refers to any chemical reaction that can occur inside of living systems without interfering with native biochemical processes Noncanonical: deviation from naturally occurring pathway; Alternative pathways HPG = l-homopropargylglycine (methionine analog) Methionine analog (HPG) carrying alkyne groups is incorporated by the cell in growing polypeptides (measures translational activity) When treated with the fluorescent dye-labeled azide, the azide group on the dye binds to the alkyne group on HPG to yield a fluorescent protein - detected 45 9/11/2024 Add a footer  I. Culture-Independent Microscopic Analyses: BONCAT- FISH A direct measure of translational activity Utilizes compounds that are synthetic molecules mimicking natural metabolites Bioorthogonal noncanonical amino acid tagging (B ONCAT) combined with FISH 46 9/11/2024 Add a footer II. Culture-Independent Genetic Analyses of Microbial Communities: Nucleic acid-based techniques 1) PCR Methods of Microbial Community Analysis 2) Microarrays for Analysis of Microbial Phylogenetic and Functional Diversity 3) Environmental Genomics and Related Methods II. Culture-Independent Genetic Analyses of Microbial Communities: 1. PCR Methods of Microbial Community Analysis Specific genes can be used as a measure of diversity Total community DNA is isolated from the habitat, and that PCR amplifies the target gene from all organisms containing the gene, which generates many copies of each gene variant, referred to as a phylotype. Techniques used in molecular biodiversity studies DNA isolation and sequencing PCR Restriction enzyme digest Electrophoresis Molecular cloning Video: PCR (Polymerase Chain Reaction) - YouTube  Molecular Aspects ❑ DNA is extracted from soil, water, blood. Can then be used as template for amplification of specific genes by PCR or as template for shotgun metagenomics. ❑ SSU rRNA analysis is used to identify community populations. DNA amplified by PCR directly from the environment. Protein-coding genes used to define phylotypes. ❑ Internal transcribed spacer region (ITS) between 16S and 23S rRNA genes may also be used. ❑ Concerns: ❑ PCR bias—certain nucleotide templates are more readily amplified than others. Steps in single-gene biodiversity analysis of a microbial community  II. Culture-Independent Genetic Analyses of Microbial Communities: 1. PCR-based Methods of Microbial Community Analysis: Uses As a preliminary step to amplify As a stand-alone diagnostic DNA for ARDRA, DGGE, SSCP or tool sequencing PRIMERS Universal or specific to a certain Specific ONLY to one organism or group group of organisms of organisms APPLICATIONS Identifying novel taxa and Identifying the presence of known community analysis organisms in the environment II. Culture-Independent Genetic Analyses of Microbial Communities: 1. PCR Methods of Microbial Community Analysis: Real Time PCR Purpose: to quantify the amount of template that is amplified by PCR (in real time) Can be used to quantify different species present in a community Need: Primers specific for the organisms you want to quantify Video: What is RT-PCR? (Real-Time PCR & Reverse Transcription PCR) - YouTube Molecular analysis of microbial Native microbial populations Communities Universal primers Targeted analysis Extract DNA Domain-specific primers PCR Kingdom-specific primers Mixed 16S rRNA Separate by cloning into E. coli Sequencing Phylogenetic identification II. Culture-Independent Genetic Analyses of Microbial Communities: DNA Fingerprints - DGGE ❑ Denaturing Gradient Gel Electrophoresis (DGGE) separates genes of the same size based on differences in base sequence Uses gradient of DNA denaturing agents (usu. formamide and urea) to separate DNA fragments. Strands melt (denature) at different denaturant concentrations DNA that would form a single band on a non-gradient gel will resolve into separate fragments (multiple bands). Limitation: Popularity has declined due to poor gel-to-gel reproducibility. II. Culture-Independent Genetic Analyses of Microbial Communities: 1. PCR Methods of Microbial Community Analysis: DGGE Bulk DN A was isolated from a microbial community and amplified by PCR using primers for 16S r R N A genes of Bacteria Six each gave a single band at the same location on the PCR gel Each band migrated to a different location on the DGGE gel DGGE study of temperature distribution of Octopus Spring cyanobacterial mat 16S rRNA variants ecotype “Who is where?”  DGGE analysis of 16S rRNA sequences Step1 : PCR Amplification Mixed Population DNAs PCR Primers Product Separate on + Denaturing 16S rRNA Gene Gradient Gel Step 2: Denaturing Gradient Gel Electrophoresis Purified Bands for Sequence Analysis Mix A B C Separation Based on Differences in Denaturant Increasing Nucleotide Sequence (G+C content) and Melting Characteristics  II. Culture-Independent Genetic Analyses of Microbial Communities: 1. PCR Methods of Microbial Community Analysis: DGGE methodology Once selective genes are amplified, PCR products are run out on an electrophoresis gel; bands are cut out and then run on a DGGE gel. The sequences are the same size as they are all the same PCR produce. However, they may differ in sequence. DGGE will separate several fragments of equal size based on sequence. Strands melt at different denaturant concentrations. Application: DGGE can be used to analyze mixed microbial samples from complex communities, such as sewage and soil. Emphasizes value of interdisciplinary approaches to ecological and microbial molecular studies II. Culture-Independent Genetic Analyses of Microbial Communities: 1. PCR Methods of Microbial Community Analysis: T-RFLP Terminal restriction fragment length polymorphism (T-RFLP): Target gene is amplified by PCR using a primer set in which one of the primers is end-labeled with a fluorescent dye Restriction enzymes are used to cut the PCR products Use: Indicates biodiversity by generating DNA fingerprints of phylotypes based on # and location of restriction sites in a specific length of DNA (e.g. 16S rRNA) METHODOLOGY: PCR with fluorescently labelled primers, so all PCR products have fluorescent terminal. Digest PCR products with restriction enzymes. Gel electrophoresis of digests Read results with a laser. The brighter the signal the more copies of DNA. Can identify individual species by checking T-RFLP pattern of pure culture. T-RFLP (Terminal-Random Fragment Length Polymorphism) PCR with fluorescently labelled primers, so all PCR products have fluorescent terminal. Digest PCR products with restriction enzymes. Run restriction digest on a gel and read with a laser. The brighter the signal the more copies of DNA. Can identify individual species by checking T- RFLP pattern of pure culture. Brightness Video: of Fluor. https://www.youtube.com/watch?v=swQ_pm4c R5o Size of fragment II. Culture-Independent Genetic Analyses of Microbial Communities: 1. PCR Methods of Microbial Community Analysis: ARISA ARISA: automated ribosomal intergenic spacer analysis Related to T-RFLP; Uses DNA sequencing Exploits the proximity of 16S and 23S rRNA genes Use: Provides detailed info of phylotype diversity by analyzing the internal transcribed spacer (ITS) region, which is length of DNA that separates 16S rRNA gene from 23S rRNA gene ITS region variable in length and base sequence in separate phylotypes. Hence diversity revealed  II. Culture-Independent Genetic Analyses of Microbial Communities: 1. PCR Methods of Microbial Community Analysis: ARISA Structure of r RNA operon spanning the 16S r R N A gene (positions 1–1540), an internal transcribed spacer (I T S) region of variable length, and the 23S r R N A gene (positions 1–2900). The P C R primers, one labeled with a fluorescent dye, are complementary to conserved sequences near the I T S region 63 9/11/2024 Add a footer  II. Culture-Independent Genetic Analyses of Microbial Communities: 1. PCR Methods of Microbial Community Analysis: Next Gen. Seq. ❑Next-generation DNA sequencers such as MINion do not require a cloning step ❑PCR products can be used directly for sequencing ❑Tremendous volume of sequence data allows for deep sequence analysis and the detection of minor phylotypes ❑Results of PCR phylogenetic analyses shows that: Several phylogenetically distinct prokaryotes are present rRNA sequences differ from those of all known laboratory cultures Molecular methods conclude that fewer than 0.1% of bacteria have been cultured and that enrichment bias is a real problem to culture-based methods  II. Culture-Independent Genetic Analyses of Microbial Communities: 1. PCR Methods of Microbial Community Analysis: Next Gen. Seq. Current sequencing platforms can generate 1012 nucleotides (nt) of sequence in a single sequencing run (requiring a week or less), with individual read lengths varying from 100 to 800 nucleotides. This enormous sequencing capacity revealed many unique phylotypes that were not detected using DGGE or clone library sequencing. 67 9/11/2024 Add a footer  II. Culture-Independent Genetic Analyses of Microbial Communities: 2. Microarrays for Analysis of Microbial Phylogenetic and Functional Diversity Microarrays are research tools with known short sequences (oligonucleotides) attached to a slide. Total community DNA is then hybridized to the slide. If the community DNA contains the same DNA, it will bind to its complement on the slide and light up. Advantage: Microarrays circumvent time-consuming steps of DGGE and T-RFLP Video: https://www.youtube.com/watch?v=0ATUjAxNf6U II. Culture-Independent Genetic Analyses of Microbial Communities: 2. Microarrays for Analysis of Microbial Phylogenetic and Functional Diversity Phylochip: microarray that focuses on phylogenetic members of microbial community Functional gene microarray (Geochip): microarray that focuses on genes of biochemical significance Advantage: Encompasses many different metabolic pathways II. Culture-Independent Genetic Analyses of Microbial Communities: 2. Microarrays for Analysis of Microbial Phylogenetic and Functional Diversity: Phylochips Specialized microarrays used to access microbial diversity in natural samples without nucleotide sequencing. Analysis of DNA hybridization reveals presence/absence of specific genes in environment. II. Culture-Independent Genetic Analyses of Microbial Communities: 2. Microarrays for Analysis of Microbial Phylogenetic and Functional Diversity: Phylochips - Methodology Thousands of genes in the microbial community can be probed for at once Method: Affix oligonucleotide probe of targeted gene to the chip surface (glass, plastic, silicon) Note position of each on the chip Isolate community DNA and amplify by PCR, fluorescently label 16S rRNA genes Add DNA to probe on the chip Fluorescence indicates hybridization to the probe and indicates presence of the specific gene  II. Culture-Independent Genetic Analyses of Microbial Communities: 2. Microarrays for Analysis of Microbial Phylogenetic and Functional Diversity: Phylochips ❑Advantages: i. Do not require cloning and sequencing, which saves time. ii. fast and cost-effective tool to detect specific microorganisms and study gene expression II. Culture-Independent Genetic Analyses of Microbial Communities: 2. Microarrays for Analysis of Microbial Phylogenetic and Functional Diversity: Phylochips Disadvantages: i. Expensive; price is not decreasing as it is with most sequencing methods. ii. May not be as specific as other methods, yielding false positives by detecting sequences that are close but not exact. iii.Though results are comparable, optimization of hybridization conditions for a large number of probes may be challenging. iv.Probe and array designing of probes and arrays time consuming.  ITRC (Interstate Technology & Regulatory Council). 2013. Team. www.itrcweb.org. ESD News and Events, 2008 II. Culture-Independent Genetic Analyses of Microbial Communities: 2. Microarrays for Analysis of Microbial Phylogenetic and Functional Diversity: Geochips  II. Culture-Independent Genetic Analyses of Microbial Communities: 3. Environmental Multi-Omics: Integration of Genomics, Transcriptomics, Proteomics, and Metabolomics ❑A more complete understanding of how a microorganism functions requires an integrated accounting of all central cellular processes. These include gene expression, functional knowledge of all gene products and product activities, and all metabolites produced during growth. ❑Multi-omics : methods of genomic, transcriptomic, proteomic, and metabolomic analysis required to unveil patterns of microbial diversity that will ultimately lead to a more predictive understanding of microbial community function and response to environmental change.  II. Culture-Independent Genetic Analyses of Microbial Communities: 3. Environmental Genomics and Related Methods Environmental genomics (metagenomics) Method: DNA is cloned from microbial community and sequenced Detects as many genes as possible Yields picture of gene pool in environment Can detect genes that are not amplified by current PCR primers Powerful tool for assessing the phylogenetic and metabolic diversity of an environment Metatranscriptomics Analyzes community RNA Reveals genes in a community that are actually expressed Reveals level of gene expression Metaproteomics This “connection graph” is intended as a visual Measures the diversity and abundance of different proteins in a representation of the complexity and abundance of community partial and complete genomes assembled from the water sample. Percentage of GC represented by strands  II. Culture-Independent Genetic Analyses of Microbial Communities: 3. Environmental Genomics and Related Methods: Metagenomics ❑ Metagenomics applied to diverse natural habitats Identification of new genes and gene products from uncultured microbes. Assembly of whole or partial genomes. Comparisons of community gene content from microbial assemblages of different origin.  II. Culture-Independent Genetic Analyses of Microbial Communities: 3. Environmental Genomics and Related Methods: Metagenomics ❑ Clone DNA fragments from environment to avoid PCR bias Predict biogeochemical conditions of a habitat based on its metagenome. ❑ mRNA and cDNA Can identify previously unidentified genes. Shows active genes. 3. Environmental Genomics and Related Methods: Single gene vs environmental genomic approaches to Microbial Community Analysis II. Culture-Independent Genetic Analyses of Microbial Communities: 3. Environmental Genomics and Related Methods: Multi-Omics Methods Overview