Marine Microbial Ecology Methods PDF

Chapter 4 Tools of Marine Microbial Ecology Methods for Studying Microorganisms in the Environment BEMA 513 – Marine microbial ecology Faculty of Science – LU Prepared by Dr Claude Daou INTRODUCTION This chapter presents the main methods for the study of marine microorganisms in...

Chapter 4 Tools of Marine Microbial Ecology Methods for Studying Microorganisms in the Environment BEMA 513 – Marine microbial ecology Faculty of Science – LU Prepared by Dr Claude Daou INTRODUCTION This chapter presents the main methods for the study of marine microorganisms in their environment. This study passes first through an adapted sampling for the marine ecosystem explored. The various components of the microbial community can then be characterized from the point of view of their biomass, their activity, and their diversity. To respond to these various issues, techniques as varied as those of flow cytometry, molecular biology, biochemistry are implemented. BEMA513_Dr Daou 2  1. Sampling Techniques in Aquatic Environment Although the development of automatic sensors now permits the in situ acquisition of many hydrological parameters related to the physiology of living organisms, water sampling still represents a primary and obligatory step in the collection of large amounts of data from the marine environment and especially for the collection of microorganisms. Collection can be considered the process of obtaining an aliquot of the studied aquatic environment. Sampling consists of retaining, preserving and storing a portion of this collected water for analytical purposes. BEMA513_Dr Daou 3  1. Sampling Techniques in Aquatic Environment → Equipments for water collection Collection of Surface Water by Hand This procedure is only applicable to collection of surface water from the water’s edge (e.g., at a beach or harbor deck) or from a small boat at a short distance from the coast. This type of sampling does not require specific equipment (the flasks that will be used to store the samples, to reduce contamination due to intermediate handling). In all cases, the hands of the sampler have to be protected by poly-ethylene gloves, and sampling is performed by immersing the bottle under the surface, as far as possible from the boat. BEMA513_Dr Daou 4 1. Sampling Techniques in Aquatic Environment → Equipments for water collection Sampling with Hydrological Bottles This technique is used to collect water at depths below the surface as well as at the surface when hand sampling is impossible or inappropriate. The Niskin bottle is currently the most commonly used device for all hydrology work. The simplest bottle is a PVC cylinder that can be hermetically sealed at both ends by removable valve systems and that have systems for attaching them to a cable. BEMA513_Dr Daou 5 1. Sampling Techniques in Aquatic Environment → Equipments for water collection Sampling with Hydrological Bottles A Niskin bottle at sea. (a) Bottle positioned at the sampling depth. Valves are open. (b) The “messenger” triggers the closure. (c) The valves are closed. The water contained in the cylinder is isolated from the outside. The bottle can then be brought on board. 6 BEMA513_Dr Daou 1. Sampling Techniques in Aquatic Environment Equipments for water collection Sampling with Hydrological Bottles Sampling rosette with 24 Niskin bottles. (a, b) Niskin bottles equipped with draw pipes, ready for sampling. (c) Rosette on the deck 7 BEMA513_Dr Daou 1. Sampling Techniques in Aquatic Environment Equipments for water collection Sampling with Hydrological Bottles Go-Flo (specific hydrological bottles that are equipped with external elastic for closure and that can be closed when immersed) on the line ready to be dropped into the water. The Go-Flo has the advantage of passing through the sea surface microlayer closed, and thereby avoids its inner surface being coating with a surface film rich in contaminants 8 BEMA513_Dr Daou 1. Sampling Techniques in Aquatic Environment Equipments for water collection Sediment Sampling The main problems posed by the study of marine sediment are associated with its heterogeneity, both horizontal and vertical. In addition, for many workers in the field of oceanography, it is necessary to recover unmixed continuous sediment samples, including sediment/water interfaces, that represent sediment fractions and surface deposits that are as little disturbed as possible. a) Octopus-type multitube corer is brought up on board. b) This is a sampler that permits simultaneous collection of 8 small sediment cores (less than 1 m in length) to study biogeochemical processes at the water-sediment interface. 9 BEMA513_Dr Daou 1. Sampling Techniques in Aquatic Environment → Equipments for water collection Sampling the Surface Microlayer (SML) of Aquatic Ecosystems The surface microlayer (SML) of aquatic ecosystems has been defined as the top 1–1,000 μm of the water surface, i.e., the interfacial region where many important bio-physicochemical processes and exchange of gases are taking place. SML plays an important role in photochemical and biologically mediated transformations of organic matter at sea surface. Depending on the type of transformations occurring in these layers, they may have important consequences in the transfer of pollutants to marine food webs. 10 BEMA513_Dr Daou 1. Sampling Techniques in Aquatic Environment Equipments for water collection Sampling the Surface Microlayer (SML) of Aquatic Ecosystems Membranes (a) To collect bacteria living in the first 10–20 μm of the SML, hydrophobic membranes are very efficient since they can collect viable bacteria by electrostatic forces. Membranes are placed on the surface of the water using a clamp. The hydrophilic Teflon membrane is often used for the isolation of bacteria because the adsorption of bacteria on the surface is lower and it provides higher rates of recovery. Techniques for sampling the surface microlayer of aquatic ecosystems. (a) Membranes; (b) rotating drum; (c) glass plate; (d) metal screen 11 BEMA513_Dr Daou  2. Sample analysis Identification of Microorganisms Phenotypic Characters ❖ Morphological ❖ Biochemical and metabolic ❖ Physiological ❖ Chemotaxonomic markers ❖ Immunological Genotypic Characters ❖ Characteristics of nucleic acids (DNA and RNA) BEMA513_Dr Daou 12 Culturing techniques BEMA513_Dr Daou 13 Cultures in Aerobiosis Bacterial colonies obtained after spreading seawater sample on different agar plates culture media and an incubation of few days. Bacterial colonies obtained from a coastal lagoon after 2 weeks of growth on R2A medium (a) and on Marine Agar 2216 medium (b).The grid on photo (c) represents 1 cm2 BEMA513_Dr Daou 14  Dioxygen Requirements and Cultures Under Anaerobic Conditions (i) Obligate aerobes that require dioxygen to grow; their respiration is aerobic. (ii) Microaerophiles, which cannot develop at dioxygen concentration equivalent to that of atmospheric level (20 %), but which still need dioxygen concentration between 2 and 10 %; their respiration is also aerobic. (iii) The facultative anaerobes, which are able to live either in the presence or in the absence of dioxygen; for example, denitrifying bacteria grow in the presence of dioxygen, but can grow in the absence of this acceptor of electrons if nitrate is available. (iv) Anaerobic air tolerant that tolerate dioxygen and grow in its presence; it is the case of some fermentative bacteria. (v) Obligate anaerobes which are inhibited or killed by dioxygen; these organisms obtain energy by fermentation or anaerobic respiration. 15 BEMA513_Dr Daou Dioxygen Requirements and Cultures Under Anaerobic Conditions Devices used for the growth of anaerobic microorganisms. (a) Vacuum chamber, the air being replaced by dinitrogen or a nitrogen- carbon dioxide. The operation is repeated 3 times; (b) Hungate tube; (c) “anaerobic” jar BEMA513_Dr Daou 16 Dilution technique BEMA513_Dr Daou 17 BEMA513_Dr Daou 18 Filtration technique BEMA513_Dr Daou 19  Microscopy technique Direct microscopic counting In direct microscopic counting: 1) dead cells are not distinguished from living cells; 2) small cells are difficult to see under the microscope; 3) precision is difficult to achieve; 4) we need a phase contrast microscope; 5) not a good method for cell suspensions of low density. BEMA513_Dr Daou 20 Microscopy technique Optical microscope examination: staining on a smear Examples : gram staining, methylene blue, Black Soudan Observation of : Shapes, sizes, mobility, grouping mode, spores, etc. BEMA513_Dr Daou 21  Microscopy technique Flow cytometry Flow cytometry is a powerful technique for the rapid analysis of single cells in a mixture. In microbiology, flow cytometry permits the reliable and rapid detection of single or multiple microbes and can provide information about their distribution within cell populations. Flow cytometry may also lead to a faster means of viability counting of microorganisms while at the same time enabling a better understanding of all bacterial cells within a given population. BEMA513_Dr Daou 22  Flow cytometry Bacillus subtilis var. Niger (BG), Escherichia coli, Micrococcus luteus and yeast cells were fixed in ethanol and stained with fluorescent dye (2.5 ug/mL fitc). A Coulter EPICS ELITE flow cytometer was used to analyze the cells immediately after adding the stain. BEMA513_Dr Daou 23 Flow cytometry BEMA513_Dr Daou 24 ❖ Biochemical characteristics 5 BEMA513_Dr Daou  ❖ Biochemical characteristics ❑ Development of API system kit The well-established method for manual microorganism identification to the species level, bioMérieux’s API identification products are test kits for identification of Gram positive and Gram negative bacteria and yeast. The system offers a large and robust database now accessible through the Internet-based APIWEB™ service. BEMA513_Dr Daou 26 ❖Biochemical characteristics The API 20E system (BioMérieux, Marcy-l'Etoile, France) is a standardized bacterial identification system that involves 21 miniaturized biochemical tests and database for species identification. It is considered one of the ‘gold standard’ methods for biochemical identification of enteric and Gram-negative bacteria in clinical, food and environmental laboratories. The biochemical tests investigated with API 20E system are: β-galactosidase (ONPG), arginine dihydrolase (ADH), lysine decarboxylase (LDC), ornithine decarboxylase (ODC), citrate utilization (CIT), H2S production (H2S), urease (URE), tryptophane deaminase (TDA), indole production (IND), Voges–Proskauer (VP), gelatinase (GEL), glucose (GLU), mannitol (MAN), inositol (INO), sorbitol (SOR), rhamnose (RHA), saccharose (SAC), melibiose (MEL), amygdalin (AMY), arabinose (ARA) and cytochrome oxidase (OX). BEMA513_Dr Daou 27 ❖Biochemical characteristics BEMA513_Dr Daou 28 ❖Biochemical characteristics BEMA513_Dr Daou 29 ❖ Chemotaxosis markers ❑ Composition and structure of the constitutive molecules of the bacterial cell  Fatty acids Components of lipids and LPS Identification of 300 different fatty acid structures Variability in the length of carbon chains, the position of double bonds, the presence of substituent groups: whole fraction analyzed Fatty acids transformed into methyl ester and separated by gas chromatography: FAME spectrum for identification BEMA513_Dr Daou 30 ❖ Chemotaxosis markers ❑ Composition and structure of the constitutive molecules of the bacterial cell  Fatty acids BEMA513_Dr Daou 31 ❖ Chemotaxosis markers Proteins Depends on the expression of the bacterial genome Electrophoretic profile of the total proteins present in the bacteria Electrophoresis in the presence of denaturing polyacrilamide: SDS-PAGE gel (Sodium Dodecyl Sulfate), migration according to their size, visualization by Coomassie blue To have a more resolving profile, a two-dimensional electrophoresis is carried out Profile compared to reference protein maps BEMA513_Dr Daou 32 The principle and method of polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE is an analytical technique to separate proteins based on their molecular weight. When proteins are separated by electrophoresis through a gel matrix, smaller proteins migrate faster due to less resistance from the gel matrix. Other influences on the rate of migration through the gel matrix include the structure and charge of the proteins. In SDS-PAGE, the use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel largely eliminates the influence of the structure and charge, and proteins are separated solely based on polypeptide chain length. SDS is a detergent with a strong protein-denaturing effect and binds to the protein backbone at a constant molar ratio. In the presence of SDS and a reducing agent that cleaves disulfide bonds critical for proper folding, proteins unfold into linear chains with negative charge proportional to the polypeptide chain length. BEMA513_Dr Daou 33 The principle and method of polyacrylamide gel electrophoresis (SDS- PAGE) 34 BEMA513_Dr Daou BEMA513_Dr Daou 35  Activity Measurement at the Cellular Level Use of Fluorescent Probes There are many molecular probes that can be used to analyze the physiological state of the cells. These probes conjugated to a fluorophore often have different targets, and they can learn about the physiological state of individual cells within a population. Fluorescent probes specific to nucleic acids are often used in microbial ecology for cell counting but also to analyze the nucleic acid content of individual cells. Within bacterial communities in aquatic environments, it is often possible to distinguish populations having different nucleic acids content. This discrimination is now often used in microbial ecology because the cells that have a high nucleic acid content are considered to be more active than those with a lower content of nucleic acids. BEMA513_Dr Daou 36 Different targets used to analyze the physiological state and identity of individual cells using fluorescent probes BEMA513_Dr Daou 37 Fluorescence in situ hybridization (FISH) FISH is a technique that uses fluorescent probes which bind to special sites of the chromosome with a high degree of sequence complementarity to the probes. The fluorescent probes are nucleic acid labeled with fluorescent groups and can bind to specific DNA/RNA sequences. Thus, we can understand where and when a specific DNA sequences exist in cells by detecting the fluorescent group. It was developed in the early 1980s. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes and flow cytometry can be used to detect the binding quantitatively. This FISH protocol is for a Cy5 and FAM labeled probe used in flow cytometry detection and fluorescence microscopy detection. BEMA513_Dr Daou 38 BEMA513_Dr Daou 39 Techniques for Microbial Diversity Studies Nucleic Acids Extraction from Environmental Samples Organisms contain DNA and RNA DNA being the cell’s long-term biological memory with a lifetime that is significantly longer than that of RNA. RNA, which is the cell’s short-term memory, is a fragile molecule that is regularly recycled to ensure responsiveness to biochemical and physical changes in the biotope. BEMA513_Dr Daou 40 Techniques for Microbial Diversity Studies The Different PCR Techniques The Regular PCR Polymerase chain reaction (PCR) method relies on the properties of DNA polymerase to extend from 5’ to 3’ end direction, an incomplete strand of a partially double- stranded DNA using the other strand as template. The double-stranded zone corresponds to the sequence on which a small fragment of about 20 nucleotides of DNA called primer can bind. This primer serves as a starting point for DNA synthesis. Using primers, it is possible to copy the two strands of a DNA fragment, with a primer binding to the sense strand (for the synthesis of the antisense strand) and the other primer binding to the antisense strand (for the synthesis and the sense strand) BEMA513_Dr Daou 41 Techniques for Microbial Diversity Studies The Different PCR Techniques The Regular PCR Amplification by PCR of a DNA fragment. Each cycle contain a denaturation, hybridization and an elongation step BEMA513_Dr Daou 42  TaqMan Real-Time Quantitative PCR The TaqMan probe-based RQ-PCR analysis exploits the 5′ → 3′ nuclease activity of the Taq polymerase to detect and quantify specific PCR products as the reaction proceeds. The internal target-specific TaqMan probe is conjugated with a reporter fluorochrome (e.g., FAM, VIC, or JOE) and a quencher fluorochrome (e.g., TAMRA). As long as these two fluorochromes are in each other's close vicinity, the fluorescence emitted by the reporter fluorochrome is absorbed by the quencher fluorochrome. However, upon amplification of the target sequence the TaqMan probe is degraded by the Taq polymerase, resulting in the separation of the reporter and quencher fluorochrome. As a result, the fluorescence signal of the reporter fluorochrome will become detectable and further increases during the consecutive PCR cycles because of the progressive accumulation of free reporter fluorochromes BEMA513_Dr Daou 43 BEMA513_Dr Daou 44 The Molecular Fingerprints Molecular fingerprinting techniques include different ways to quickly view and analyze diversity between gene fragments amplified by PCR. The most common are the RFLP (T-RFLP) and PFGE BEMA513_Dr Daou 45  PFGE Pulsed-field gel electrophoresis (PFGE) is a laboratory technique used by scientists to produce a DNA fingerprint for a bacterial isolate. The procedure for this technique is relatively similar to performing a standard gel electrophoresis except that instead of constantly running the voltage in one direction, the voltage is periodically switched among three directions; one that runs through the central axis of the gel and two that run at an angle of 120 degrees either side. The pulse times are equal for each direction resulting in a net forward migration of the DNA. This procedure takes longer than normal gel electrophoresis due to the size of the fragments being resolved and the fact that the DNA does not move in a straight line through the gel. BEMA513_Dr Daou 46 BEMA513_Dr Daou 47 BEMA513_Dr Daou 48  The Molecular Fingerprints RFLP (Restriction Fragment Length Polymorphism) This method permits to analyze sequence diversity of PCR fragments after their hydrolysis with restriction enzymes and determination of the size of the hydrolyzed products by electrophoresis. The chosen enzymes generally recognize a sequence of four nucleotides and on a statistic point of view are enzymes that cut frequently DNA. The resolution of the technique will depend on the enzyme used and for a given study, different tests must initially be performed. BEMA513_Dr Daou 49 Technique RFLP BEMA513_Dr Daou 50 RFLP BEMA513_Dr Daou 51  Technique RFLP RFLP and T-RFLP techniques. PCR amplified fragments are hydrolyzed with a restriction enzyme (1). The length of generated fragments (A, B, C, D) is visualized by electrophoresis (2). For RFLP, all fragments are stained, whereas for T-RFLP, only the terminal fragments (A and C) are visualized by fluorescence since during PCR, one of the two primers harbored a fluorochrome (yellow spot) 52  The Molecular Fingerprints RFLP (Restriction Fragment Length Polymorphism) The multitude of bands can increase the difficulty of analysis and variations of this technique have been proposed. To limit the number of bands after electrophoresis, one of the fragments is analyzed by the method called T-RFLP (Terminal Restriction Fragment Length Polymorphism). For this, one of the terminal moiety of the PCR fragment is detected by fluorescence because during PCR, one of the two primers used included a fluorochrome group. BEMA513_Dr Daou 53  The Molecular Fingerprints ARDRA (amplified rDNA restriction analysis) If the RFLP and T-RFLP are applicable to any gene, the ARDRA analysis method (ARDRA amplified rDNA restriction analysis) focuses on the diversity of ribosomal genes only. The primers and enzymes are standardized allowing the identification of organisms thanks to a data bank containing size of the fragments generated corresponding to different reference bacterial strains. BEMA513_Dr Daou 54 RIBOPRINTER BEMA513_Dr Daou 55 Riboprinter BEMA513_Dr Daou 56  Figure 2 ARDRA profiles of various isolates Lane L represents the 100 bp molecular marker and all the other lanes Figure 1 Dendrogram showing SSU rRNA gene of all correspond to thermophilic bacterial the isolates obtained from Northern Ireland in isolates used. relation to known authentic sequences of Geobacilli BEMA513_Dr Daou 57