Methods in Microbial Ecology

Transcription

1 LECTURE PRESENTATIONS For BROCK BIOLOGY OF MICROORGANISMS, THIRTEENTH EDITION Michael T. Madigan, John M. Martinko, David A. Stahl, David P. Clark Chapter 22 Lectures by John Zamora Middle Tennessee State University Methods in Microbial Ecology 2012 Pearson Education, Inc. I. Culture-Dependent Analyses of Microbial Communities 22.1 Enrichment 22.2 Isolation 1

2 22.1 Enrichment Isolation The separation of individual organisms from the mixed community Enrichment Cultures Select for desired organisms through manipulation of medium and incubation conditions Inocula (singular Inoculum) The sample from which microorganisms will be isolated Figure 22.1 The isolation of Azotobacter. 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 NH 4 plate Selection for aerobic N 2 -fixing bacteria usually results in the isolation of Azotobacter or its relatives. By contrast, enrichment with fixed forms of nitrogen such as NH 4 + rarely results in isolating nitrogen-fixing bacteria because there is no selective pressure for nitrogen fixation. 2

3 22.1 Enrichment Enrichment Cultures Can prove the presence of an organism in a habitat Cannot prove an organism does not inhabit an environment The ability to isolate an organism from an environment says nothing about its ecological significance Animation: Enrichment Cultures 2012 Pearson Education, Inc. Marmara University Enve303 Env. Eng. Microbiology Prof. BARIŞ ÇALLI 22.1 Enrichment The Winogradsky Column An artificial microbial ecosystem (Figure 22.2) Serves as a long-term source of bacteria for enrichment cultures Named for Sergei Winogradsky First used in late 19th century to study soil microorganisms 3

4 Figure 22.2 The Winogradsky column. 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 column is incubated in a location that receives subdued sunlight. Anoxic decomposition leading to sulfate reduction creates the gradient of H 2 S. Thiospirillum jenense (purple sulfur bacteria) Chromatium okenii (purple sulfur bacteria) Chlorobium limicola (green sulfur bacteria) Photo of Winogradsky columns that have remained anoxic up to the top; each column had a bloom of a different phototrophic bacterium Enrichment 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 4

5 22.2 Isolation Pure cultures contain a single kind of microorganism Can be obtained by streak plate, agar shake, or liquid dilution (Figure 22.3) Agar dilution tubes are mixed cultures diluted in molten agar Useful for purifying anaerobic organisms Figure 22.3 Pure culture methods Organisms that form distinct colonies on plates are usually easy to purify. Colonies Paraffin mineral oil seal Colonies of phototrophic purple bacteria in agar dilution tubes; the molten agar was cooled to app. 45 o C before inoculation. A dilution series was established from left to right, eventually yielding wellisolated colonies. The tubes were sealed with a 1:1 mixture of sterile paraffin and mineral oil to maintain anaerobiosis 5

6 22.2 Isolation Most-probable-number technique Serial 10 dilutions of inocula in a liquid media Used to estimate number of microorganisms in food, wastewater and other samples (Figure 22.4) Animation: Serial Dilutions and a Most Probable Number Analysis 2012 Pearson Education, Inc. Marmara University Enve303 Env. Eng. Microbiology Prof. BARIŞ ÇALLI Figure 22.4 Procedure for a most-probable-number (MPN) analysis 1 ml (liquid) or 1 g (solid) Enrichment culture or natural sample Dilution 1 ml 1 ml 1 ml 1 ml 1 ml Growth Growth No growth 9 ml of broth 1/10 (10 1 )

7 22.2 Isolation Axenic culture (grown under sterile conditions) can be verified by Microscopy Observation of colony characteristics Tests of the culture for growth in other media Laser tweezers are useful for Isolating slow-growing bacteria from mixed cultures (Figure 22.5) Figure 22.5 The laser tweezers for the isolation of single cells b a a F b F a Cell b Beam focus Objective lens of microscope A laser beam strongly focused on a very small object such as a cell creates downward radiation forces that allow the cell to be dragged in any direction. Laser beam Optically trapped cell Capillary tube Laser Severing point Mixture of cells The laser beam can lock onto a single cell present in a mixture in a capillary tube and drag the optically trapped cell away from the other cells. Once the desired cell is far enough away from the other cells, the capillary is severed and the cell is flushed into a tube of sterile medium. 7

8 22.2 Isolation Flow cytometry Uses lasers Suspended cultures passed through specialized detector Cells separated based on fluorescence II. Culture-Independent Analyses of Microbial Communities 22.3 General Staining Methods 22.4 Fluorescent In Situ Hybridization (FISH) 22.5 PCR Methods of Microbial Community Analysis 22.6 Microarrays and Microbial Diversity: Phylochips 22.7 Environmental Genomics and Related Methods 8

9 22.3 General Staining Methods Fluorescent staining using DAPI or acridine orange (AO) DAPI-stained cells fluoresce bright blue (Figure 22.6a) AO-stained cells fluoresce orange or greenish-orange (Figure 22.6b) DAPI and AO fluoresce under UV light DAPI and AO are used for the enumeration of microorganisms in samples DAPI and AO are nonspecific and stain nucleic acids Cannot differentiate between live and dead cells Figure 22.6 Nonspecific fluorescent stains (a) DAPI and (b) acridine orange (AO) staining showing microbial communities inhabiting activated sludge in a municipal wastewater treatment plant. With acridine orange, cells containing low RNA levels stain green. (c) SYBR Green stained sample of Puget Sound (Washington, USA) surface water showing green-fluorescing bacterial cells. The large cells near the center of the field are m in diameter 9

10 22.3 General Staining Methods Viability stains: differentiate between live and dead cells (Figure 22.7) Two dyes are used Based on integrity of cell membrane Green cells are live Red cells are dead Can have issues with nonspecific staining in environmental samples Figure 22.7 Viability staining Live (green) and dead (red) cells of Micrococcus luteus (cocci) and Bacillus cereus (rods) stained by the LIVE/DEAD BacLight Bacterial Viability Stain 10

11 22.3 General Staining Methods Fluorescent antibodies can be used as a cell tag Highly specific Making antibodies is time consuming and expensive Green fluorescent protein can be genetically engineered into cells to make them autofluorescent Can be used to track bacteria Can act as a reporter gene 22.4 Fluorescent In Situ Hybridization (FISH) Nucleic acid probe is DNA or RNA complementary to a sequence in a target gene or RNA FISH: fluorescent in situ hybridization (Figure 22.9) Phylogenetics of microbial populations (Figure 22.10) Used in microbial ecology, food industry, and clinical diagnostics CARD-FISH 11

12 Figure 22.9 Morphology and genetic diversity The photomicrographs shown here, produced by (a) phase-contrast and (b) a technique called phylogenetic FISH, are of the same field of cells. Although the large oval cells are of a rather unusual morphology and size for prokaryotic cells and all look similar in phase-contrast microscopy, the phylogenetic stains reveal that there are two genetically distinct types (one stains yellow and one stains blue). Figure FISH analysis of sewage sludge (a) Nitrifying bacteria. Red, ammonia-oxidizing bacteria; green, nitrite-oxidizing bacteria. (b) Confocal laser scanning micrograph of a sewage sludge sample. The sample was treated with three phylogenetic FISH probes, each containing a fluorescent dye (green, red, or purple) that identifies a particular group of Proteobacteria. Green-, red- or purplestained cells reacted with only a single probe; other cells reacted with multiple probes to give blue or yellow. 12

13 22.4 Fluorescent In Situ Hybridization (FISH) CARD-FISH FISH can be used to measure gene expression in organisms in a natural sample (Figure 22.11) A FISH method that enhances the signal is called catalyzed reporter deposition FISH (CARD-FISH) Figure Catalyzed reporter deposition FISH (CARD-FISH) labeling of Archaea Archaeal cells in this preparation fluoresce intensely (green) relative to DAPIstained cells (blue) 13

14 22.5 PCR Methods of Microbial Community Analysis Specific genes can be used as a measure of diversity Techniques used in molecular biodiversity studies (Figure 22.12) DNA isolation and sequencing PCR Restriction enzyme digest Electrophoresis Molecular cloning Figure Steps in single-gene biodiversity analysis of a microbial community Microbial community Amplify by PCR using fluorescently tagged primers Restriction enzyme digest and run on gel PCR DNA Sample Gel Extract total community DNA Amplify 16S RNA genes using general primers (for example, Bacteria-specific) or more restrictive primers (to target endospore-forming Bacteria) T-RFLP gel Sample All 16S rrna genes Sample Excise bands and clone 16S rrna genes Different 16S rrna genes DGGE gel Excise bands Sequence Generate tree from results using endosporespecific primers Bacillus subtilis Env 1 Bacillus cereus Bacillus megaterium Env 2 Clostridium histolyticum Env 3 Sequence Generate tree from results using endosporespecific primers 14

15 22.5 PCR Methods of Microbial Community Analysis DGGE: denaturing gradient gel electrophoresis separates genes of the same size based on differences in base sequence (Figure 22.13) Denaturant is a mixture of urea and formamide Strands melt at different denaturant concentrations Figure PCR and DGGE gels PCR amplification DGGE 15

16 22.5 PCR Methods of Microbial Community Analysis T-RFLP: terminal restriction fragment length polymorphism Target gene is amplified by PCR Restriction enzymes are used to cut the PCR products ARISA: automated ribosomal intergenic spacer analysis Related to T-RFLP Uses DNA sequencing 22.5 PCR Methods of Microbial Community Analysis Results of PCR phylogenetic analyses Several phylogenetically distinct prokaryotes are present rrna sequences differ from those of all known laboratory cultures Molecular methods conclude that less than 0.1% of bacteria have been cultured 16

17 22.6 Microarrays and Microbial Diversity: Phylochips Phylochip: microarray that focuses on phylogenetic members of microbial community (Figure 22.15) Circumvents time-consuming steps of DGGE and T-RFLP Figure Phylochip analysis of sulfate-reducing bacteria diversity Positive Weak positive Negative Each spot on the microarray shown has an oligonucleotide complementary to a sequence in the 16S rrna of a different species of sulfate-reducing bacteria. After the microarray is hybridized with 16S rrna genes PCR amplified from a microbial community and then fluorescently labeled, the presence or absence of each species is signaled by fluorescence (positive or weak positive) or nonfluorescence (negative), respectively. 17

18 22.7 Environmental Genomics and Related Methods Environmental genomics (metagenomics) 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 III. Measuring Microbial Activities in Nature 22.8 Chemical Assays, Radioisotopic Methods, and Microelectrodes 22.9 Stable Isotopes Linking Specific Genes and Functions to Specific Organisms 18

19 H 2 35 S Lactate or H 2 S 11/27/ Chemical Assays, Radioisotopes, & Microelectrodes In many studies, direct chemical measurements are sufficient (Figure 22.18) Higher sensitivity can be achieved with radioisotopes Proper killed cell controls must be used Figure Microbial activity measurements Chemical measurement: Lactate and H 2 S transformations during sulfate reduction. Sulfate reduction Formalin-killed control H 2 S Lactate 14 CO2 incorporation Photosynthesis Light Killed Dark Radioisotopic measurement: photosynthesis measured with 14 CO 2 Time Time Radioisotopic measurement: sulfate reduction measured with 35 SO 4 2- Sulfate reduction H 2 present Killed H 2 absent 14 CO2 evolution 14 C-Glucose respiration Radioisotopic measurement production of 14 CO 2 from 14 C- glucose. Killed Time Time 19

20 22.8 Chemical Assays, Radioisotopes, & Microelectrodes Microelectrodes Can measure a wide range of activity ph, oxygen, CO 2, and others can be measured Small glass electrodes, quite fragile (Figure 22.19) Electrodes are carefully inserted into the habitat (e.g., microbial mats) Measurements taken every mm (Figure 22.20) Figure Microelectrodes Gold Glass Platinum Schematic drawing of an O 2 microelectrode. 5 mm The platinum rod functions as a cathode and when voltage is applied, O 2 is reduced to H 2 O, generating a current. The current resulting from the reduction of O 2 at the gold surface of the cathode is proportional to the O 2 concentration in the sample. Note the scale of the electrode. Membranes Glass mm NO 3 N 2 O 2e + N 2 O H 2 O N 2 2 OH Bacteria Cathode Nutrient solution Biological microsensor for the detection of nitrate (NO 3- ). Bacteria immobilized at the sensor tip denitrify NO 3 - (or NO 2- ) to N 2 O, which is detected by reduction to N 2 at the cathode 20

21 Depth in sediment (mm) 11/27/2012 Figure Depth profiles of oxygen and nitrate. Data obtained using the lander equipped with microelectrode sensors for remote chemical characterization of deep-sea sediments. Oxygen (O 2 ) concentration (mm) Seawater 0 O 2 Oxic sediment NO Anoxic sediment Nitrate (NO 3 ) concentration (mm) Figure Deployment of deepsea lander. The lander is equipped with a bank of microelectrodes to measure distribution of chemicals in marine sediments Stable Isotopes Nonradioactive isotopes of an element Used to study microbial transformations in nature Isotope fractionation Carbon and sulfur are commonly used Lighter isotope is incorporated preferentially over heavy isotope (Figure 22.22) 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 21

22 Figure Mechanism of isotopic fractionation with carbon as an example Enzyme substrates Enzyme that fixes CO 2 Fixed carbon 12 CO 2 12 C organic 13 CO 2 13 C organic Enzymes that fix CO 2 preferentially fix the lighter isotope ( 12 C). This results in fixed carbon being enriched in 12 C and depleted in 13 C relative to the starting substrate. The size of the arrows indicates the relative abundance of each isotope of carbon Linking Specific Genes and Functions to Specific Organisms Flow cytometry and multiparametric analysis Natural communities contain large populations Flow cytometer examines specific cell parameters very fast (Figure 22.26) Cell size Cell shape Fluorescence Parameters can be combined and analyzed (multiparametric analysis) 22

23 Figure Flow cytometric cell sorting 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) As the fluid stream exits the nozzle, it is broken into droplets containing no more than a single cell. Droplets containing desired cell types (detected by fluorescence or light scatter) are charged and collected by redirection into collection tubes by positively or negatively charged deflection plates Linking Specific Genes and Functions to Specific Organisms Radioisotopes used as measures of microbial activity in a microscopic technique called microautoradiography (MAR) Radioisotopes can also be used with FISH FISH microautoradiography (FISH-MAR) Combines phylogeny with activity of cells (Figure 22.27) 23

24 Figure Fluorescent in situ hybridization (FISH) combined with microautoradiography (MAR). An uncultured filamentous cell belonging to the Gammaproteobacteria is shown to be an autotroph (as revealed by MAR-measured uptake of 14 CO 2 ). Uptake of 14 C-glucose by a mixed culture of Escherichia coli (yellow cells) and Herpetosiphon aurantiacus (filamentous, green cells). MAR of the same field of cells shown in (b). Incorporated radioactivity exposes the film and shows that glucose was assimilated mainly by cells of E. coli 24