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Applied and Environmental Microbiology, September 2006, p. 5948-5956, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00696-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Application of the [3H]Leucine Incorporation Technique for Quantification of Bacterial Secondary Production Associated with Decaying Wetland Plant Litter

Jane E. Gillies, Kevin A. Kuehn,* Steven N. Francoeur, and Robert K. Neely

Department of Biology, Eastern Michigan University, Ypsilanti, Michigan 48197

Received 26 March 2006/ Accepted 26 June 2006

The radiolabeled leucine incorporation technique for quantifying rates of bacterial production has increased in popularity since its original description for bacterioplankton communities. Prior studies addressing incorporation conditions (e.g., substrate saturation) for bacterial communities in other habitats, such as decaying plant litter, have reported a wide range of final leucine concentrations (400 nM to 50 µM) required to achieve saturation-level uptake. We assessed the application of the [3H]leucine incorporation procedure for measuring bacterial production on decaying wetland plant litter. Substrate saturation experiments (nine concentrations, 10 nM to 50 µM final leucine concentration) were conducted on three dates for microbial communities colonizing the submerged litter of three emergent plant species (Typha angustifolia, Schoenoplectus validus, and Phragmites australis). A modified [3H]leucine protocol was developed by coupling previously described incubation and alkaline extraction protocols with microdialysis (500 molecular weight cutoff membrane) of the final radiolabeled protein extract. The incorporation of [3H]leucine into protein exhibited a biphasic saturation curve, with lower apparent Km values ranging from 400 nM to 4.2 µM depending on the plant species studied. Upper apparent Km values ranged from 1.3 to 59 µM. These results suggest differential uptake by litter-associated microbial assemblages, with the lower apparent Km values possibly representing bacterial uptake and higher apparent Km values representing a combination of both bacterial and nonbacterial (e.g., eukaryotic) uptake.

* Corresponding author. Present address: Department of Biological Sciences, 118 College Dr., #5018, The University of Southern Mississippi, Hattiesburg, MS 39406-0001. Phone: (601) 266-4748. Fax: (601) 266-5797. E-mail: kevinkuehn{at}usm.edu.

Applied and Environmental Microbiology, September 2006, p. 5948-5956, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00696-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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