TY - JOUR

T1 - A new method for determining the metabolic activity of specific bacterial populations in soil using tritiated leucine and immunomagnetic separation

AU - Sengeløv, Gitte

AU - Sørensen, Søren Johannes

AU - Frette, Lone

AU - Kroer, Niels

N1 - Key words Immunomagnetic separation - Tritiated leucine - Metabolic activity - Microbial soil communities - Pseudomonas putida

PY - 2000

Y1 - 2000

N2 - A new assay, using immunomagnetic separation and uptake of tritiated leucine ([3H]-Leu), was developed for measuring the in situ metabolic activity of specific bacterial populations in soil. Such assays are needed to assess the role individual species play in diverse microbial soil communities. The method was optimized using Pseudomonas putida KT2440¿:¿:Tc+/TOL::gfp inoculated into soil microcosms. Inoculated soil samples were incubated with [3H]-Leu followed by an immunomagnetic separation to recover the target bacteria. Radiolabel incorporated by the target bacteria was then measured. Incubation time with immunomagnetic beads was not critical for optimal target cell recovery, but samples needed to be washed at least 5¿times during the immunomagnetic separation to reduce unspecific binding of the indigenous soil bacteria to the magnetic beads. Soil absorption of the polyclonal antibody further reduced this unspecific binding, resulting in <1% contamination by indigenous soil bacteria relative to numbers of recovered target cells. The assay was tested by investigating the effect of different incubation temperatures on the metabolic activity of the target cells. As expected, a linear relationship between activity and temperature was observed, demonstrating the sensitivity of the assay. The method was applied to compare activities of the target strain in bulk soil and in the rhizosphere of barley. Contrary to what was anticipated, no significant difference in metabolic activity was observed.

AB - A new assay, using immunomagnetic separation and uptake of tritiated leucine ([3H]-Leu), was developed for measuring the in situ metabolic activity of specific bacterial populations in soil. Such assays are needed to assess the role individual species play in diverse microbial soil communities. The method was optimized using Pseudomonas putida KT2440¿:¿:Tc+/TOL::gfp inoculated into soil microcosms. Inoculated soil samples were incubated with [3H]-Leu followed by an immunomagnetic separation to recover the target bacteria. Radiolabel incorporated by the target bacteria was then measured. Incubation time with immunomagnetic beads was not critical for optimal target cell recovery, but samples needed to be washed at least 5¿times during the immunomagnetic separation to reduce unspecific binding of the indigenous soil bacteria to the magnetic beads. Soil absorption of the polyclonal antibody further reduced this unspecific binding, resulting in <1% contamination by indigenous soil bacteria relative to numbers of recovered target cells. The assay was tested by investigating the effect of different incubation temperatures on the metabolic activity of the target cells. As expected, a linear relationship between activity and temperature was observed, demonstrating the sensitivity of the assay. The method was applied to compare activities of the target strain in bulk soil and in the rhizosphere of barley. Contrary to what was anticipated, no significant difference in metabolic activity was observed.

U2 - 10.1007/s003740000236

DO - 10.1007/s003740000236

M3 - Journal article

VL - 32

SP - 202

EP - 208

JO - Biology and Fertility of Soils

JF - Biology and Fertility of Soils

SN - 0178-2762

IS - 3

ER -