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Monitorización de GEMs en el ambiente. Marcadores.

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Presentation on theme: "Monitorización de GEMs en el ambiente. Marcadores."— Presentation transcript:

1 Monitorización de GEMs en el ambiente. Marcadores

2 Why monitor domesticated microbial inoculants in nature? Risk assessment of GMMs Performance/behaviour studies- Ag/Biotech. applications –Biological pesticides –Bioremediation –Biological fertilizers (Rhizobia) Basic studies of microbial ecology

3 Questions to address: How many cells are present? Are the cells alive? Are the cells metabolically active? How are the cells distributed? Can the cells perform their intended tasks? What effect do the cells have on the natural microbial diversity?

4 Molecular Probes Marker Genes

5 Marker genes as specific monitoring tools- I - XylE protein (Catecol 2-3 dioxigenase) - LacZ protein (  -Galactosidase) Impredictability (inactivated by O 2 …) Well studied and widely used Activity absent in Pseudomonadaceae Different substrates: X-Gal, ONPG, MUG Background activity Visible only in big amounts of cells (colonies) Detection of life cells

6 - LacZ protein - XylE protein

7 - GFP (gfp): Enumerate total cell population Regardless of physiological status Detect by fluorescence-based methods - Flow cytometry - Fluorescence microscopy - Firefly luciferase (luc) or bacterial luciferase (luxAB) Monitor metabolically active cells in the population Detect light emission - Luminometry - Microscopy + sensitive cameras Marker genes as specific monitoring tools- II

8 Bioluminiscencia 1. Origen eucariótico (genes luc luciérnaga) LH 2 + ATP + O 2 CO 2 + oxiluciferina + AMP+ luz Mg 2+ luciferasa 2. Origen bacteriano (genes lux Vibrio / Photobacterium) FMNH 2 + RCHO + O 2 H 2 O + ROOH + FMN + luz Mg 2+ luciferasa

9 Bioluminiscence luxCDABE AB code for the luciferase CDE code for luciferin biosynthesis Strategies: Introduce the whole operon Constitutively luminescent bacteria ~8kb operon, interference with FA biosynthesis Introduce the luciferase Luciferin has to be externally added Reaction always depends on reducing power -> cell status

10 Fluorescencia Green fluorescent protein (GFP de Aequorea victoria) Fluorescencia verde al excitarse con luz UV o azul- sin sustrato ni cofactor

11 Luminometry (lux-tagged cells) Flow cytometry (gfp-tagged cells) gfp/luxAB-tagged bacteria Nycodenz density gradient Bacterial fraction Cryosection Confocal microscopy Fluorescence stereomicoscopy

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13 P. fluorescens SBW25 in soil

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15 Confrontation studies with antagonistic fungal strains Trichoderma harzianum - GFP

16 Marker Genes: monitorisation of E. Coli-GFP colonisation in whole animals

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18 E.coli-GFP infecting peritoneal cavity

19 Molecular Probes Marker Genes

20 Molecular probes to detect GEMs Immunological techniques DNA probes PCR-based methods

21 Immunological techniques - Fluorescent microscopy (single cells) - ELISA (>100 cells) Advantages: Highest specificity (serotyping) Detection at single-cell stage Drawbacks: Cross-reaction Auto-fluorescence Epitope expression

22 Rhizobium sp.Bradirhizobium sp.

23 DNA probes - Taxonomic probes - Phylogenetic probes Advantages: Taxonomic level specificity Sensitivity of 16S probes Direct detection of interesting activities Drawbacks: Specificity > species level Crossreaction (diversity unknown)

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25 16S RNA

26 Fluorescence in situ hybridization (FISH)

27 FISH Taxonomic probes In situ hybridization of a vertical biofilm slice with a NIT3-labeled probe specific for the genus Nitrobacter (red stain cluster) correlated to oxygen and nitrate gradients measured by microelectrodes.

28 FISH Functional probes Confocal microscopic image of a bacterial aggregate thin section after hybridization with a Cy3-labeled probe specific for nitrite- oxidizing Nitrospira sp. (red) and a Cy5- labeled probe specific for ammonia- oxidizing Nitrosospira sp. (blue). 20 µm

29 PCR-based methods - PCR --> RFLP Advantages: Highest sensitivity (1 cell/gr.) In situ detection of activity Drawbacks: Inspecificity Contamination Interference of humic substances Alterations due to sample purification

30 Total soil DNA Restriction digestion PCR 16S rRNA genes Eubacterial primers 5´primer fluorescent Separation on sequencing gel T-RFLP (Terminal-Restriction Fragment Length Polymorphism)


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