Presentation on theme: "Monitorización de GEMs en el ambiente. Marcadores."— Presentation 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?
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
- 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) 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
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
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.
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
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
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)