Quantification of cell-specific substrate uptake by probe-defined bacteria under in situ conditions by microautoradiography and fluorescence in situ hybridization.
A technique based on quantitative microautoradiography (QMAR) and fluorescence in situ hybridization (FISH) was developed and evaluated in order to determine the quantitative uptake of specific substrates in probe-defined filamentous bacteria directly in a complex system. The technique, QMAR-FISH, has a resolution of a single cell and is based on an improved fixation protocol and the use of an internal standard of bacteria with known specific radioactivity. The method was used to study the in situ ecophysiology of the filamentous bacteria 'Candidatus Meganema perideroedes' and Thiothrix sp. directly in an activated sludge system. The cellular uptake rate of tritium-labelled substrates revealed an average cell-specific uptake rate of 4.1 yen 10-15 mol of acetate cell-1 h-1 and 3.1 yen 10-15 mol of acetate cell-1 h-1 for the two filamentous species respectively. The two filamentous species had very similar activity in all cells along each filament. Surprisingly, the filaments within both probe-defined populations had threefold variation in activity between the different filaments, demonstrating a large variation in activity level within a single population in a complex system. The substrate affinity (Ks) for uptake of acetate of the cells within the two filamentous bacteria was determined by incubation with variable concentrations of labelled acetate. The Ks values of the 'Candidatus Meganema perideroedes' and the Thiothrix filamentous bacteria were determined to be 1.8 micro M and 2.4 micro M acetate respectively.
Nielsen
Jeppe Lund
JL
Section of Environmental Engineering, Aalborg University, Sohngaardsholmsvej 57, DK-9000 Aalborg, Denmark. jeppe@bio.auc.dk
Christensen
Dinna
D
Kloppenborg
Marie
M
Nielsen
Per Halkjaer
PH
D000085:Acetates / Q000378:metabolism*; D020561:Alphaproteobacteria / Q000145:classification* / Q000235:genetics / Q000378:metabolism*; D001345:Autoradiography; D002250:Carbon Radioisotopes / Q000378:metabolism; D020563:Gammaproteobacteria / Q000145:classification* / Q000235:genetics / Q000378:metabolism*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D018613:Microscopy, Confocal; D015345:Oligonucleotide Probes; D012722:Sewage / Q000382:microbiology*; D014316:Tritium / Q000378:metabolism; D014865:Waste Disposal, Fluid
D000085:Acetates; D002250:Carbon Radioisotopes; D015345:Oligonucleotide Probes; D012722:Sewage; D014316:Tritium