MICR3213_Methods in Microbial Ecology Part 2_ 2024.pdf

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 ...

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