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 usedActivity absent in Pseudomonadaceae Different substrates: X-Gal, ONPG, MUG

Background activityVisible 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

Bioluminiscencia1. 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

BioluminiscenceluxCDABE 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)