Technical considerations for the use of 15N-DNA stable-isotope probing for functional microbial activity in soils.
Stable-isotope DNA probing is a culture-independent technique that may provide a link between function and phylogeny of active microorganisms. The technique has been used in association with 13C substrates while here we evaluate feasibility and limitations of 15N-DNA stable-isotope probing (SIP) using labelled and unlabelled pure microbial cultures or soil extracts. Our results showed that (15)N-DNA probing is feasible for cultures as well as soil samples. Limitations of 15N-DNA-SIP are (a) the need for relatively large quantities of DNA to visualise bands (although molecular resolution is much higher) and (b) 15N-DNA enrichment needed to ideally be >50 at%; however, this requirement can be lowered to approx. 40 atom% 15N with pure cultures using a modified CsCl centrifugation method (140K g for 69 h). These advances in 15N-DNA-SIP methodology open new opportunities to trace active microbial populations utilising specific N substrates in situ.
Cadisch
Georg
G
Institute of Plant Production and Agroecology in the Tropics and Subtropics (380a), University of Hohenheim, 70593 Stuttgart, Germany. cadisch@uni-hohenheim.de
Espana
Mingrelia
M
Causey
Rachel
R
Richter
Michael
M
Shaw
Eve
E
Morgan
J Alun W
JA
Rahn
Clive
C
Bending
Gary D
GD
D002499:Centrifugation, Density Gradient; D002586:Cesium; D002712:Chlorides; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D009587:Nitrogen Isotopes; D016958:Pseudomonas putida / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012987:Soil / Q000032:analysis*; D012988:Soil Microbiology
D002712:Chlorides; D004269:DNA, Bacterial; D009587:Nitrogen Isotopes; D012987:Soil; D002586:Cesium; C028019:cesium chloride