MICR3213_Methods in Microbial Ecology Part 1_ 2024.pdf

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, mo...

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