Lecture 11 - Environmental Microbiology PDF

BIOL371: Microbiology Lecture 11 – Environmental microbiology 1 Topics of today 1. Culture-dependent analyses of microbial communities 2. Culture-independent microscopic analyses of microbial communities 3. Culture-independent molecular analyses of microbial communities 4. Measuring microbial ac...

BIOL371: Microbiology Lecture 11 – Environmental microbiology 1 Topics of today 1. Culture-dependent analyses of microbial communities 2. Culture-independent microscopic analyses of microbial communities 3. Culture-independent molecular analyses of microbial communities 4. Measuring microbial activities in nature Materials covered:  Chapters 19.1-19.11  Figures 19.1, 19.4-19.11, 19.13, 19.16, 19.19, 19.20, 19.23, 19.25, 19.29, 19.31, 19.35, 19.36, 19.38, 19.42 2 Enrichment culture microbiology  Inoculum: sample from which microorganisms will be isolated  Isolation: separation of individual populations from the mixed community  Enrichment cultures: used to isolate bacteria from diverse and densely populated samples; e.g., feces or soil  Select for desired organisms through manipulation of medium and incubation conditions  Favouring growth of target organisms while inhibiting growth of non-target organisms  Example: isolation of the aerobic nitrogenfixing bacterium Azotobacter 3 Enrichment culture outcomes  Successful enrichment cultures are those with appropriate resources (nutrients) and conditions (pH, temperature, osmotic pressure etc) that are needed for target organisms to grow  Enrichment cultures can show that the presence of an organism in a habitat  Cannot rule out that an organism does not inhabit an environment  Say nothing about the relative abundance and ecological importance of the target organism  Enrichment bias: microorganisms cultured in the lab are often minor components of the microbial ecosystem  Quantity of nutrients available in the lab are typically much higher than encountered in nature  Dilution of inoculum maybe used to eliminate rapidly growing, but quantitatively insignificant species 4 Classical procedures for isolating microorganisms  Most-probable-number technique:  Serial 10X dilutions of inoculum in a liquid medium  Often used to estimate number of microorganisms in food, wastewater, and other samples  Pure (axenic) culture need to be verified  Colony characteristics  Microscopy on cell shape, single morphological type, uniform staining (e.g., Gram stain)  Tests multiple culture conditions for expected growth and no growth 5 Laser tweezers for isolating single cells  Laser tweezers: Laser beams move through the objective lens of a microscopy  Focus on the cell and separate it from other cells  Isolate slow-growing bacteria from mixed cultures 6 Flow cytometry  Method of counting and examining a mixtures of cells  Stream of suspended cells passing through an electronic detector in single file  Cells sorted by cell shape, size, or fluorescent properties 7 High-throughput cultivation of previously uncultured microorganisms  Separates individual cells for culture in microtitre plate, one cell per well  Each cell can grow without competition from other species  Important in isolating slow growing species that thrive in nutrient poor environments 8 Microfluidic platform for cultivation  Microfabrication to construct tiny wells for cultivation, one cell per well 9 General staining to examine microbial communities  Culture-independent microscopic method of examining microbial communities  Commonly used nonspecific fluorescent dyes – fluoresce under ultraviolet light  DAPI (4’,6-diamidino-2-phenylindole) – blue  Acridine orange – orange  SYBR Green – green  Nonspecific stains for nucleic acids, cannot distinguish live from dead cells 10 Viability stains  Two dyes used  Green dye enters both dead or live cells  Red dye (propidium iodide) can enter only dead cells (defective cytoplasmic membrane)  Provides information on abundance and viability 11 Fluorescent protein reporters  Genes encoding fluorescent proteins controlled by bacterial gene promoters  Introduced into bacteria to track live bacteria and bacterial processes (e.g., infection) Green fluorescent protein Cells of Sinorhizobium meliloti (arrows) carrying a plasmid with an α-galactoside-inducible promoter fused to the green fluorescent protein gene; the cells are on clover seedling roots. Green fluorescence indicates that α -galactosides are released and available to support the growth of this bacterium 12 General principle of nucleic acid hybridization  Nucleic acids with complementary sequence can form hybrid (DNA:DNA, DNA:RNA, or RNA:RNA)  The probe should be single-stranded nucleic acid  Fluorescently labelled for most experiments  Following hybridization, the unbound probes are removed by washing with buffer  Multiple probes, each labelled with a different colour fluorescent dye and complementary to a specific organism or group of organisms, can be used to examine populations of microorganisms 13 Fluorescence in situ hybridization (FISH)  In situ means “in place”: used to see microorganisms in the context of their communities  Fluorescence in situ hybridization (FISH): the probing nucleic acid is tagged with a fluorescent dye and used to hybridize with nucleic acids of microorganisms in the community Phase contrast micrograph Same image as (a) visualized with phylogenetic FISH to three different groups of bacteria 14 Phylogenetics of microbial populations with FISH  Fluorescently labelled oligonucleotides complementary to rRNA  Can be highly specific to species, or multiple species or genera  Used in microbial ecology, food industry, and clinical diagnostics  Can be modified to measure gene expression or translational activity Confocal laser scanning micrograph of a sewage sludge sample treated with different phylogenetic FISH probes 15 PCR methods of microbial community analysis  Specific genes can be used as a measure of diversity  Isolate DNA from environmental samples  PCR (polymerase chain reaction) amplification of specific genes; typically rRNA genes  Analysis of the amplified genes  Molecular cloning  Electrophoresis  Restriction enzyme digestion  Sequencing 16 Phylogenetic analysis: massively parallel DNA sequencing  Multiple sequencing technologies available  Do not require molecular cloning  Generate billions of sequence reads  Allows the detection of minor phylotypes  Results of phylogenetic analysis  rRNA sequences differ from those of all known laboratory cultures  New phylogenetic distinct prokaryotes  Fewer than 0.1% of bacteria have been cultured, enrichment bias a real problem in environmental microbiology 17 Geochip – functional gene microarray The image shows green fluorescence of varying intensity, approximating gene abundance. The red spots correspond to repeated applications of a known amount of a reference DNA standard  DNA microarray containing gene probes that encompass most major biogeochemical processes  Fluorescently labelled environmental DNA and hybridize to Geochip  Relatively fast and easy to analyze  Provides functional information to correlate with phylogenetic analysis Functional category Gene families Total probes Carbon cycling 149 26922 Nitrogen cycling 32 6493 Sulfur cycling 27 4739 Phosphorus 7 3260 Metal homeostasis 121 43432 Viruses 115 2857 Other 81 10380 Organic remediation 104 11591 Virulence 639 21152 Secondary metabolism 68 4032 Electron transfer 15 797 Stress response 89 26306 1447 161,961 Total 18 Environmental multi-omics  A more complete understanding of how a microorganism functions requires an integrated accounting of all central cellular processes  These include integrative knowledge on:  Genomics – genes, gene function, and gene regulation  Transcriptomics – global gene expression under different conditions  Proteomics – accumulation of proteins under different conditions  Metabolomics – dynamics of accumulation of metabolites  Expand the analysis to community level: metagenomics, metatranscriptomics, metaproteomics, and metametabolomics 19 Environmental genomics (metagenomics)  DNA extracted from environmental samples  Sequence, assemble and annotate – mostly partial genomes assembled  Identify as many genes as possible  Detect genes not amplified by available PCR primers  Powerful tool for assessing the phylogenetic and metabolic diversity of an environment 20 Metatranscriptomics  Analysis of community mRNA  Remove rRNA before sequencing (>90% of total RNA are rRNA)  Reveals genes in a community that are active  Reveals level of gene expression Metatranscriptomic analysis of coastal marine surface waters 21 Metaproteomics and metabolomics  Metaproteomics: measures the diversity and abundance of different proteins in a community  Current state of technology can only detect the most abundant proteins  Metabolomics: the comprehensive analysis of cellular and extracellular metabolites of a microbial community 22 Direct chemical measurements of metabolites  For many studies, direct chemical measurements are sufficient; e.g., lactate and H2S can be measured with high sensitivity by chemical assay  For some processes, higher sensitivity can be achieved with radioisotopes Chemical measurements of lactate and H2S transformations during SO42− reduction Radioisotopic measurement of photosynthesis as measured with 14CO2 23 Microsensors  Microsenors available to measure a wide range of activity; e.g., pH, oxygen  Small glass electrodes that can be inserted into the habitat Oxygen (O2) microsensor. Oxygen diffuses through the silicone membrane in the microsensor tip and reacts with electrons on the gold surface of the cathode, forming hydroxide ions (OH−); the latter generates a current proportional to the O2 concentration in the sample. Biological microsensor for the detection of nitrate (NO3−). Bacteria immobilized at the sensor tip denitrify NO3− or NO2− to N2O, which is detected by electrochemical reduction to N2 at the cathode. 24 Isotopic fractionation  Stable isotopes: an element can have multiple nonradioactive stable isotopes; e.g., 12C and 13C  Isotopic fractionation: biological reactions prefer lighter isotopes; hence cellular materials are enriched in 12C and depleted in 13C relative to inorganic carbon  The ratio of 12C and 13C can be used to trace the biological or geological origin of ancient environment Isotopic fractionation Isotopic geochemistry:  Petroleum (derived from plants) displays similar isotopic fractionation as plants  Marine carbonate is of geological origin 25 Stable isotope probing  Stable isotope probing: feed microorganisms with substrate labelled with stable heavy isotope; e.g., 13C-benzoate  Microorganisms that can utilize benzoate will incorporate 13C into their DNA  The heavier DNA can be separated by ultracentrifugation  Compare the 13C-labelled DNA sequence with metagenomics data will identify microorganisms that can utilize benzoate in the microbial community 26