monitorización de gems en el ambiente. marcadores
TRANSCRIPT
Monitorización de GEMs en el ambiente.
Marcadores
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
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?
Molecular Probes
Marker GenesMarker Genes
Marker genes as specific monitoring tools- I
- XylE protein (Catecol 2-3 dioxigenase)
- LacZ protein (-Galactosidase)
Impredictability (inactivated by O2…)
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
- LacZ protein
- XylE protein
- 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
Bioluminiscencia
1. Origen eucariótico (genes luc luciérnaga)
LH2 + ATP + O2 CO2 + oxiluciferina + AMP+ luzMg2+
luciferasa
2. Origen bacteriano (genes lux Vibrio / Photobacterium)
FMNH2 + RCHO + O2 H2O + ROOH + FMN + luzMg2+
luciferasa
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
Fluorescencia
Green fluorescent protein (GFP de Aequorea victoria)
Fluorescencia verde al excitarse con luz UV o azul- sin sustrato ni cofactor
Luminometry (lux-tagged cells)
Flow cytometry (gfp-tagged cells)
gfp/luxAB-tagged bacteria
Nycodenz density gradient
Bacterial fraction
Cryosection
Confocal microscopy
COLOR CCD(FLUORESCENCE)DIGITAL CCD(LUMINESCENCE)Fluorescence stereomicoscopy
COLOR CCD(FLUORESCENCE)DIGITAL CCD(LUMINESCENCE)
P. fluorescens SBW25 in soil
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Time (Days)
Confrontation studies with antagonistic fungal strains Trichoderma harzianum - GFP
Marker Genes: monitorisation of E.
Coli-GFP colonisation in whole animals
E.coli-GFP infecting peritoneal cavity
Molecular Probes
Marker Genes
Molecular probes to detect GEMs
Immunological techniques
DNA probes
PCR-based methods
Immunological techniques
- Fluorescent microscopy (single cells)
- ELISA (>100 cells)
Advantages:Highest specificity (serotyping)Detection at single-cell stage
Drawbacks:Cross-reaction Auto-fluorescenceEpitope expression
Rhizobium sp. Bradirhizobium sp.
DNA probes
- Taxonomic probes- Phylogenetic probes
Advantages:Taxonomic level specificitySensitivity of 16S probesDirect detection of interesting activities
Drawbacks:Specificity > species levelCrossreaction (diversity unknown)
16S RNA
Fluorescence in situ hybridization (FISH)
FISHTaxonomic
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.
FISHFunctional
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
PCR-based methods
- PCR --> RFLP
Advantages:Highest sensitivity (1 cell/gr.)In situ detection of activity
Drawbacks:InspecificityContaminationInterference of humic substancesAlterations due to sample purification
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)