Microbiology 微生物学 Spring-Summer

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2 Microbiology 微生物学 2017 Spring-Summer

3 Relevant Information and Resources Course slides can be found at 教学工作 Course-related questions will be answered through s. Textbook: Brock Biology of Microorganisms (13 th )

4 Chapter 23 Microbial Ecosystems I Microbial Ecology

5 Ecological Concepts Ecosystem: the sum total of all of the organisms and abiotic factors in a particular environment. (a) Taihu, China (b) St. John s River, USA Normal (high richness) (a) St. John s River, USA Abnormal (low richness but high abundance)

6 Ecological Concepts Microbial species diversity: Richness (Total number of different species) vs. Abundance (The proportion of each species in the community)

7 Microbial Ecosystems

8 Microbial Ecosystems Light Community 1 Community 2 Photic zone: Oxic zone: Oxygenic phototrophs Aerobes and facultative 6 CO 2 6 H 2 O aerobes C 6 H 12 O 6 6 O 2 C 6 H 12 O 6 6 O 2 6 CO 2 6 H 2 O Energy yield Community 3 Anoxic sediments: 1. Guild 1: denitrifying bacteria (NO 3 N 2 ) ferric iron-reducing bacteria (Fe 3 Fe 2 ) 2. Guild 2: sulfate-reducing bacteria (SO 2 4 H 2 S) sulfur-reducing bacteria (S 0 H 2 S) 3. Guild 3: fermentative bacteria 4. Guild 4: methanogens (CO 2 CH 4 ) acetogens (CO 2 acetate) Populations, Guilds (metabolically similar microbial populations that exploit the same resources in a similar way), and Communities

9 Microbial Ecosystems Human activities CO 2 Respiration Land plants Animals and microorganisms Fossil fuels Humus Soil formation Aquatic plants and phytoplankton CO 2 Biological pump Death and mineralization Aquatic animals CO 2 Earth s crust Rock formation Biogeochemical Cycling: CO 2

10 Microbial Ecosystems (CH 2 O) n Biogeochemical Cycling: C Organic matter Oxygenic photosynthesis Chemolithotrophy Respiration Methanotrophy CO 2 Oxic Anoxic Methanogenesis Syntroph assisted Acetogenesis Anoxygenic photosynthesis Anaerobic respiration and fermentation Organic matter (CH 2 O) n

11 Microbial Ecosystems Biogeochemical Cycling: N Nitrification groups of protein Nitrogen fixation DRNA groups of protein Nitrogen fixation Oxic Anoxic Anammox Denitrification

12 Microbial Ecosystems Ferrous iron oxidation (bacterial or chemical) (Ferric) Ferric iron reduction (bacterial or chemical) Biogeochemical Cycling: Fe Smelting of ores Chemical oxidation (Ferrous)

13 Distance (mm) Microbial Environment Microorganisms, Niches (prime vs. fundamental), Microenvironments Nutrient levels and growth rates: feastor-famine; Competition vs. Cooperation Distance (mm) Oxygen microenvironments in a small soil particle

14 Surface and Biofilms Greater access to nutrients, protection, staying in a favorable habitat Microcolonies Root Microorganisms on surface a). A plant root, b) a microscopic slide immersed in a river

15 Surface and Biofilms Biofilms: Assemblages of bacterial cells attached to a surface and enclosed in an adhesive matrix secreted by the cells. Cells in biofilm Surface a). A cross-sectional view of a biofilm; b) a natural biofilm by Confocal microscope; c) A natural biofilm on a rock.

16 Biofilm formation Attachment Colonization Development (adhesion of a (intercellular (more growth and few cells to a suitable solid surface) communication, growth, and polysaccharide formation) polysaccharide) Cell O Water channels Surface Polysaccharide

17 Biofilm constituents and Regulation Extracellular substances: Polysaccharide, Protein, DNA c-di-gmp Regulatory factor: c-di-gmp

18 Biofilm control Methods: 1) To Prevent from forming; 2) To Induce dispersion

19 Chapter 22 Methods in Microbial Ecology I Culture-Dependent Analysis of Microbial Communities

20 Enrichment Enrichment: 1. Inocula; 2. Medium; 3. Conditions; 4. Techniques Enrichment Bias Mineral salts medium containing mannitol but lacking NH 4, NO 3, or organic nitrogen. NH 4 Soil Incubate aerobically +NH 4 plate NH 4 plate +NH 4 plate Isolation of Azotobacter NH 4 plate

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23 Enrichment

24 Enrichment Lake or pond water Mud supplemented with organic nutrients and CaSO 4 Gradients Column O 2 H 2 S Foil cap Algae and cyanobacteria Purple nonsulfur bacteria Sulfur chemolithotrophs Patches of purple sulfur or green sulfur bacteria Anoxic decomposition and sulfate reduction The Winogradsky Column

25 Isolation Pure culture methods Colonies Paraffin seal

26 Isolation Isolation: 1. The streak plate; 2. Agar dilution tubes; 3. Most-Probable Number (MPN); Purity verification: 1. Microscopy; 2. Colonies; 1 ml (liquid) or 1 g (solid) 9-ml broth Growth Enrichment culture or natural sample Dilution 1 ml 1 ml 1 ml 1 ml 1 ml Growth No growth 1/10 (10 1 ) Other media; Procedure for MPN analysis

27 Isolation b a a b Cell Beam focus Objective lens of microscope Laser beam Optically trapped cell Severing point Capillary tube Laser Mixture of cells The laser tweezers for the isolation of single cells

28 Chapter 22 Methods in Microbial Ecology II Culture-Independent Analysis of Microbial Communities

29 Staining Nonspecific fluorescent stains Nucleic acid staining: Viable & Dead DAPI Acridine orange DAPI (4,6 -diamido-2- phenylindole SYBR Green I Non-Specific fluorescent strains

30 Staining Viability staining: Dye unable to penetrate membrane Using with Propidium iodide nucleic acid staining (penetrating membrane) Live Green; Dead -- Red

31 Staining Fluorescent Antibodies: Antigen Bacterial cell Antibacterial antibody, labeled with fluorescent dye Fluorescent antibodies as a cell tag Direct immunofluorescence Method ONE

32 Staining Fluorescent Antibodies: Antigen Bacterial cell Antibacterial antibody (unlabeled) made in rabbit Method TWO Anti-rabbit Ig, labeled with fluorescent dye Indirect immunofluorescence

33 Staining Green Fluorescent Protein (GFP)

34 FISH Fluorescence in situ hybridization Phylogenetic Staining using FISH

35 FISH Phylogenetic Staining using FISH Sewage sludge

36 FISH CARD (catalyzed reporter deposition) -- FISH Probe + peroxidase : tyramide fluorescent intermediate + protein

37 PCR-based method Steps in single gene biodiversity analysis Fhylotype: the microbial diversity of a given habitat

38 PCR-based method DGGE: Denaturing Gradient Gel Electrophore sis

39 PCR-based method

40 PCR-based method Fhylotype: the microbial diversity of a given habitat T-RFLP: Terminal Restriction Fragment Length Polymorphism

41 Fluorescence PCR-based method Position S rrna gene Forward PCR primer containing fluorescent tag ( ) ITS region bp Position 1513 Position 23 23S rrna gene Reverse PCR primer Community DNA PCR Gel analysis Fragment size (base pairs) ARISA: Automated Ribosomal Intergenic Spacer Analysis.

42 Chips Positive Weak positive Negative Phylochips

43 Environmental Genomics Microbial community Extract DNA Community sampling approach Total community DNA Environmental genomics approach Amplify single gene, for example, gene encoding 16S rrna Sequence and generate tree Restriction digest total DNA and then shotgun sequence Assembly and annotation Outcomes Genomes Single gene phylogenetic tree 1. Phylogenetic snapshot of most members of the community 2. Identification of novel phylotypes Total gene pool of the community 1. Identification of all gene categories 2. Discovery of new genes 3. Linking genes to phylotypes

44 Percent identity Fold coverage Environmental Genomics Genomic islands 100 ISL1 ISL2 ISL3 ISL4 ISL5 80 Fold coverage Position along the Prochlorococcus genome (kbp) Metagenomic analysis: Comparison of the genome sequences of cultured strains with Prochlorococcus genes obtained from metagenomic analyses of ocean water.

45 Microbial Activity in Nature Microbial Activity Measurements by Chemical Assays using Radioisotopes and Microelectrodes Sulfate reduction Formalin-killed control Time Sulfate reduction H 2 S Lactate 14 CO2 incorporation Photosynthesis Light Killed Dark Time 14 C-Glucose respiration H 2 present Killed H 2 absent Killed Time Time

46 Microbial Activity in Nature Gold Glass Platinum O 2 Microelectrode 5 m Membranes Glass m NO 3 N 2 O 2e 1 N 2 O H 2 O N 2 2 OH Bacteria Cathode Nutrient solution NO 3 - Microelectrode

47 Depth in sediment (mm) Microbial Activity in Nature Oxygen (O 2 ) concentration ( M) O 2 Seawater Oxic sediment NO 3 Microbial Activity Measurements: Depth profiles of oxygen and nitrate in deepsea sediments 10 Anoxic sediment Nitrate (NO 3 ) concentration ( M)

48 Microbial Activity in Nature Enzyme substrates Stable Isotopes Enzyme that fixes CO 2 Fixed carbon Isotopic Fractionation: Enzymes typically favor the lighter isotope. These discrimination between isotopes are the result of biological activities.

49 Microbial Activity in Nature Marine carbonate Calvin cycle plants Atmospheric CO 2 Methane Petroleum Isotopic Geochemistry of 13 C and 12 C Cyanobacteria Purple sulfur bacteria Green sulfur bacteria Recent marine sediments 3.5 billion-year-old rocks C ( 0 / 00 )

50 Microbial Activity in Nature Isotopic Geochemistry of 34 S and 32 S Igneous rocks Marine sulfate Sedimentary sulfide Meteoritic sulfide Lunar sulfides Sulfide from marine mud Elemental sulfur S ( 0 / 00 )

51 13 C ( 0 / 00 ) Microbial Activity in Nature SIMS: Secondary Ion Mass Spectrometry able to work on a single cell so detected activity can be assigned to specific microbes Ion source Primary ions Sample Mass spectrometer Magnet ABCDE Multiple detectors Secondary ions Outer shell of sulfate-reducing bacteria cells (green) burned away by primary ion beam Inner core of methanotrophic Archaea cells (red) exposed Cycles of sputtering and data collection (increasing depth into aggregate)

52 Microbial Activity in Nature Flow Cytometry: Cells labeled by FISH Sample stream Nozzle Light scatter and fluorescence detector Laser Deflection plates Induces charge on selected droplets Sorted samples Waste (non labeled cells)

53 Microbial Activity in Nature FISH-MAR: MicroAuto- Radiography

54 Colin Murrell Microbial Activity in Nature This cell metabolizes 13 C substrate 13 C-DNA 12 C-DNA Environmental sample Feed 13 C substrate These cells do not metabolize 13 C substrate Extract DNA 12 C-DNA Separate light (12C) from heavy ( 13 C) DNA 13 C-DNA Remove and analyze (PCR 16S rrna or metabolic genes, or do genomics) Ultracentrifuge tube with DNA SIP: Stable Isotope Probing

55 Microbial Activity in Nature Single-Cell Genomics: Label cells by FISH Isolate fluorescent cells by flow cytometry Extract DNA PCR DNA Multiple displacement amplification (MDA) and sequencing Primers Phage DNA polymerase MDA: Multiple Displacement Amplification Assay for specific genes (16S rrna genes, metabolic genes, etc.) A B C Sequence genome