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)