This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Gillies, J. E. Articles by Neely, R. K. Search for Related Content PubMed PubMed Citation Articles by Gillies, J. E. Articles by Neely, R. K. Agricola Articles by Gillies, J. E. Articles by Neely, R. K.

 Previous Article  |  Next Article 

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

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

Received 26 March 2006/ Accepted 26 June 2006

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 K_m values ranging from 400 nM to 4.2 µM depending on the plant species studied. Upper apparent K_m values ranged from 1.3 to 59 µM. These results suggest differential uptake by litter-associated microbial assemblages, with the lower apparent K_m values possibly representing bacterial uptake and higher apparent K_m values representing a combination of both bacterial and nonbacterial (e.g., eukaryotic) uptake.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

 
Freshwater marshes and lake littoral zones are widely recognized as among the most productive ecosystems on earth (31). Emergent vascular plants, such as Typha (cattail) and Phragmites (reed), often comprise a major portion of the plant biomass produced within these habitats (see, for example, references 33 and 35). Most of this living plant biomass enters the pool of decaying plant material after plant senescence and death (6), where microorganisms (bacteria and fungi) play an important role in its decomposition and mineralization (3, 7, 17, 19, 20). Hence, given the detrital-based nature of most wetland ecosystems, quantifying the conversion and flow of carbon and nutrients from plant detritus into associated microbial decomposers (bacteria and fungi) would seem to be a pivotal component in our understanding energy flow and elemental cycling within these systems.

In aquatic ecosystems, bacteria are widely regarded as playing an important role in organic matter processing (3, 5, 25). The use of techniques for estimating rates of bacterial secondary production from rates of incorporation of radiolabeled substrates (i.e., thymidine and leucine) have increased since their original description and have provided important quantitative information concerning the functional role of bacteria in carbon cycling within both freshwater and marine ecosystems. Originally developed for estimating production rates of planktonic bacteria in oceanic environments (9, 15), both the thymidine and the leucine methods have been modified for their application in other aquatic habitats, such as freshwater pelagic zones (2, 14, 23), sediments (2, 8, 10, 16, 29), and biofilms associated with decaying plant litter (2, 26, 27, 28, 34). Modifications of the original methods for their application to other ‘nonplanktonic’ habitats have often developed from a critical assessment of the assumptions underlying the radiotracer approach (e.g., substrate specificity, substrate saturation, linearity of incorporation, and efficiency of extraction of the radiolabeled macromolecule) (see, for example, references 2, 8, 26, and 27).

Early studies examining the efficacy of the [³H]leucine incorporation technique for quantifying rates of bacterial production associated with decaying plant matter in freshwater ecosystems have reported that saturation of leucine incorporations rates occurred at a leucine concentration of 400 nM (26, 27), which was ca. 20 to 40 times higher than saturation concentrations observed for planktonic bacteria in marine waters (9, 15). A modification to the labeled protein extraction technique that involved an initial ultrasonication step prior to the standard trichloroacetic acid (TCA) protein precipitation and filtration was assumed to effectively remove litter-attached bacteria, and hence radiolabeled bacterial protein, from the decaying litter substratum (see, for example, reference 27).

Buesing and Gessner (2), using an improved alkaline protein extraction method, observed in a recent study that saturation of leucine incorporation rates for bacterial communities associated with decaying Phragmites australis litter occurred at a leucine concentration nearly 2 orders of magnitude higher than previously reported (50 µM versus 400 nM), suggesting that (i) prior extraction protocols (i.e., ultrasonication alone) were not effective in removing radiolabeled protein from the litter substrate and (ii) the functional role of bacteria in carbon transformations within aquatic ecosystems (i.e., littoral wetlands) may be significantly underestimated (3). In their method description, Buesing and Gessner (2) solubilized proteins associated with litter samples using an alkaline solution after initial ultrasonication, centrifugation, and washing procedures to removed unincorporated [³H]leucine. However, despite the more efficient extraction of radiolabeled protein, high background levels in killed controls (i.e., signal/noise ratio of 4:1) suggested the absorption of free [³H]leucine radiolabel to litter samples, which could potentially compromise the accurate determination of leucine incorporation rates. In addition, Buesing and Gessner (2) advised that caution must be used when litter-associated fungal biomass greatly exceeds (ca. >20 times) bacterial biomass, since there is potential for fungal uptake of [³H]leucine when final leucine concentrations are within the micromolar range.

The main objective of the present study was to further examine the major assumptions (i.e., substrate saturation and linearity) underlying the [³H]leucine incorporation technique for estimating the productivity of bacterial communities associated with submerged decaying wetland plant matter. We sought to expand on the work reported recently by Buesing and Gessner (2) by assessing saturation of [³H]leucine incorporation rates for bacterial assemblages colonizing the decaying litter of three different wetland plant species (Typha angustifolia, Schoenoplectus validus, and P. australis). In addition, we conducted saturation experiments on three occasions during bacterial development (2, 4, and 8 weeks) on plant litter in order to assess potential differences in litter from different plant species and possible changes in the saturation of [³H]leucine incorporation rates during bacterial colonization and development. In an attempt to improve the resolution in saturation uptake kinetics of [³H]leucine incorporation into protein, these saturation uptake experiments included a wider range of leucine concentrations (nine total) than those tested earlier (six total) by Buesing and Gessner (2). Additional studies also examined the linearity of leucine incorporation into protein. Finally, we used an alkaline protein extraction method coupled with microdialysis (500 molecular weight cutoff membrane [MWCO]), instead of ultrasonication and centrifugation, to help facilitate the removal of "free" unincorporated radiolabel while ensuring the retention of radiolabeled macromolecules (i.e., protein).

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Study site.
This study was conducted in a small (18.2-ha) inland emergent marsh wetland, Paint Creek, located in southeast Michigan (N42°12.971, W083°37.181). The study site is a created retention basin wetland formed more than 20 years ago and receives waters draining the surrounding Paint Creek watershed. The study was situated in the southeastern corner of the wetland that is dominated by the emergent macrophytes T. angustifolia and S. validus, as well as P. australis.

Field procedures.
Overwintered standing-dead litters of T. angustifolia (leaves) and S. validus (culms), and P. australis (leaves) were collected (Spring 2004) from the Paint Creek wetland, returned to the laboratory, air dried, and stored at ambient lab temperatures until used. Air-dried litter was cut into ca. 10-cm-long sections, and approximately 3 g of litter was enclosed in ca. 35-by-15-cm plastic fine (1-mm) mesh litter bags. Sixty litter bags containing corresponding litter from each plant species were submersed in the wetland surface waters (20-cm depth). Three replicate litter bags of each species were randomly retrieved after 2, 4, and 8 weeks (saturation experiments conducted in June, July, and August 2004) or 3 weeks (linearity experiments conducted in April 2005). Collected litter bags were gently enclosed in resealable containers with wetland water, placed on ice in a cooler, and returned to the laboratory (<30 min). In the laboratory, litter was removed from the litter bags and cut into either 1.7-cm-long pieces (T. angustifolia and S. validus) or circular leaf discs (5 cm², P. australis) for all subsequent experimental analyses (see below).

Leucine incorporation. (i) Substrate saturation and isotope dilution.
Two replicate litter sections (T. angustifolia and S. validus) or four leaf discs (P. australis) from corresponding litter bags were placed into sterile (autoclaved), 20-ml glass scintillation vials containing 4 ml of 0.2-µm-pore-size filtered wetland water (final incubation volume), with fixed amounts of L-[4,5-³H]leucine (TRK683 Amersham; specific activity, 5.6 TBq/mmol) and increasing amounts of unlabeled L-leucine (Sigma Corp.). Three replicates and two TCA-killed controls (5% [vol/vol] final concentration) were used for each of nine leucine concentrations ranging from 0.01 to 50 µM (specific activity = 1.1 GBq to 5.6 TBq/mmol). Incubations were conducted at ambient laboratory temperatures (ca. 20°C) for 30 min. To reduce the potential for autodegradation of the leucine radiolabel, concentrated (185 MBq/ml) high-specific-activity [³H]leucine (ca. 5.6 TBq/mmol) obtained from the manufacturer was immediately diluted by using a sterile technique to ca. 12 MBq/ml in 2% ethanol. This stock [³H]leucine material was stored at 4°C (never frozen) and used for incorporation experiments within 2 months.

After incubation, leucine incorporation was stopped by the addition of 440 µl of 50% TCA (5% [vol/vol] final concentration). Sample vials were incubated in a circulating water bath at 80°C for 30 min, cooled to room temperature, and placed on ice for 30 min to precipitate proteins. The resultant precipitated proteins were vacuum filtered through mixed nitrocellulose filters (25 mm, 0.22 µm, Millipore Corp.) using a 0.8-µm mixed nitrocellulose backing filter. Filters and litter pieces were washed three times each with 4 ml of cold 5% TCA and then washed two times each with 4 ml of cold 80% ethanol. Filters and litter pieces were then washed two times each with 4 ml of cold sterile distilled water and placed into 15-ml polypropylene conical centrifuge tubes (Corning, Inc.).

Next, 10 ml of alkaline extractant (0.3 N NaOH, 0.1% sodium dodecyl sulfate [SDS], 25 mM EDTA) was added to each tube, and the tubes were incubated at 80°C in a dry-block heater for 60 min. The tubes were removed and cooled to room temperature. Sample aliquots (500 µl) of extracts were placed into sterile 1.8-ml plastic microcentrifuge tubes and adjusted to a pH 7 with the addition of HCl. Sample aliquots (100 µl) of this solution were placed into individual wells of a Microdialyser System 100 (part 0066315; Pierce Chemical, Inc.) and dialyzed overnight (12 h) against an ammonium bicarbonate buffer (0.2 M NH₄HCO₃, 0.1% SDS, 25 mM EDTA, 0.1 M NaCl) through a 500 MWCO dialysis membrane (Spectrum, Inc.). After dialysis, liquid samples were removed, mixed with 100 µl of 50% H₂O₂ in 20-ml glass scintillation vials, and incubated overnight to clear samples of humic coloration (see reference 32). Subsequently, 10 ml of scintillation fluid (Ecolume) was added to each vial, mixed, and allowed to sit for >24 h before the radioactivity was determined by liquid scintillation using a Beckman LS6500 liquid scintillation counter.

The rates of leucine incorporation were fitted to the hyperbolic function of Michaelis-Menten-type kinetics by using iterative least-squares nonlinear regression analysis (Systat 10.2). The fitted parameters were used to estimate V_max (maximum leucine incorporation rates) and the apparent K_m (i.e., K_t + S_n) for each saturation experiment. Isotope dilution was determined as the ratio of V_max to V_measured (V_meas) at leucine concentrations, where saturation uptake rates were observed (see references 2 and 8).

(ii) Linearity of incorporation.
Time course experiments were conducted at a leucine concentration at which initial saturation of [³H]leucine uptake was observed (i.e., 2.5 µM). For linearity experiments, two replicate litter sections (T. angustifolia and S. validus) or four leaf discs (P. australis) were incubated at ambient laboratory temperatures (ca. 20°C) in 20-ml glass scintillation vials with 4 ml of 0.2-µm-pore-size filtered wetland water containing 2.5 µM [4,5-³H]leucine (specific activity = 22 GBq mmol^–1). Three replicates were used for each of six time intervals ranging from 0 to 90 min. After incubation, leucine incorporation was stopped by the addition of 440 µl of 50% TCA (5% final concentration), and radiolabeled protein was extracted and quantified according to the procedures described above. Rates of [³H]leucine incorporation into protein were fitted to a linear regression model (Systat 10.2).

Bacterial biomass.
Bacterial biomass associated with litter samples was determined by epifluorescence microscopy after staining with SYBR Gold I (Molecular Probes, Inc.) (see reference 21). Two replicate litter sections (T. angustifolia and S. validus) or four leaf discs (P. australis) from corresponding litter bags were placed into sterile (autoclaved), 20-ml glass scintillation vials containing 10 ml of a 2% phosphate-buffered (0.1% sodium pyrophosphate) formalin. Bacterial cells attached to litter were detached by probe ultrasonication (50 W; Cole-Parmer, Inc.) for 1 min on ice (1). After ultrasonication, samples were gently mixed, and aliquots (30 to 200 µl) placed into a 25-mm glass vacuum filtering apparatus containing 2 ml of filtered (0.2 µm pore size) sterile distilled water. An additional 2 ml of sterile distilled water was added to ensure a homogenous suspension of bacterial cells prior to filtration. Samples were then vacuum (20 kPa) filtered through 25-mm supported Anodisc filters (25 mm, 0.2 µm pore size; Whatman), with a 0.8-µm-pore-size mixed cellulose-ester membrane backing filters (Fisher Scientific, Inc).

Filters were removed from the filtering apparatus and placed sample side up on separate drops of 2.5% (vol/vol, diluted from stock) SYBR Gold I in clean plastic petri dishes and stained for 15 min in darkness. Residual moisture from filters was removed after staining by resting the backside of the filters on Kimwipes. Filters were mounted on glass slides with 30 µl of antifade mounting solution containing 50% glycerol, 50% phosphate-buffered saline (120 mM NaCl, 10 mM NaH₂PO₄ [pH 7.5]), and 0.1% p-phenylenediamine (21). A 25-mm circular glass coverslip was then placed on the filter surface, and a drop of immersion oil (Cargille, type DF, formula 1261) was applied. Bacterial cells were enumerated (at x1,000 magnification) in a minimum of 10 fields (350 cells) by using a Leica DMRB epifluorescence microscope, and cells were assigned into six categories according to size and shape. Video capture photographs were taken of representative bacterial cells by using a DEI-470 video camera system (Optronics Engineering, Inc.), and corresponding bacterial size classes were measured by using Scion Image analysis software (Scion Corp.) (see reference 12). Biovolume estimates (µm³) for each size class were calculated from length (l) and width (w) measurements according to the following formula: V = (1/4) · w² · (l – w) · + (1/6) · w³ · . Biovolume estimates were converted to bacterial carbon (fg C) using a formula (C = 89.5 · V ^0.59), which accounts for size-dependent differences in carbon density of bacterial cells (24).

Fungal biomass.
Fungal biomass associated with collected litter samples was estimated from concentrations of a fungal membrane sterol, ergosterol (11). Two replicate litter sections (T. angustifolia and S. validus) or four leaf discs (P. australis) were placed in sterile (autoclaved) 20-ml glass scintillation vials and stored frozen at –20°C until analyzed. Frozen samples were lyophilized and weighed, and ergosterol was extracted in alcoholic KOH (0.8% KOH in high-pressure liquid chromatography-grade methanol; total extraction volume, 10 ml) for 30 min at 80°C in tightly capped digestion tubes with constant stirring. The resultant crude extract was partitioned into n-pentane and evaporated to dryness in 15-ml glass conical vials under a stream of nitrogen gas (18). Ergosterol in dried samples was redissolved by ultrasonication (10 min) in 1 ml of methanol and centrifuged, and the supernatants were stored tightly capped 2-ml screw cap vials at –20°C in darkness until analyzed. A LichroSpher 100 RP-18 column (0.46 by 25 cm; Merck, Inc.) maintained in a Shimadzu column oven (CTO-10AS) at 40°C and connected to a Shimadzu autosampler (SIL-10AD) and a Shimadzu liquid chromatograph system (pump LC-10AT, controller SCL-10A) were used for separation and analysis. The mobile phase was high-pressure liquid chromatography-grade methanol at a flow rate of 1.5 ml min^–1. Ergosterol was detected at 282 nm by using a Shimadzu (SPD-10A) UV/VIS detector (retention time of ca. 8 min) and was identified and quantified based on comparison with ergosterol standards (Fluka Chemical Co.). For the determination of litter-associated fungal biomass, litter ergosterol concentrations were converted to fungal carbon, assuming a conversion factor of 10 µg of ergosterol mg^–1 fungal C and 43% C in fungal dry mass (11).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Leucine incorporation: substrate saturation and isotope dilution.
Leucine incorporation rates into protein exhibited biphasic saturation curves, with rates of leucine incorporation reaching initial saturation at concentrations below ca. 5 µM and a second saturation plateau occurring at concentrations greater than ca. 10 µM (Fig. 1 to 3). A similar biphasic response was observed for all plant litter types, and incubation times (i.e., 2, 4, and 8 weeks) were examined. For T. angustifolia, the initial saturation of leucine incorporation rates were relatively consistent, occurring at a final leucine concentration of 2.5 µM throughout bacterial development on decaying litter (Fig. 1). In contrast, leucine concentrations needed for the initial saturation of the leucine incorporation rates associated with S. validus and P. australis litter decreased during bacterial development (Fig. 2 and 3). During early bacterial development (2 weeks), saturation of leucine incorporation rates was observed at a final concentration of 2.5 µM. However, during later bacterial development (8 weeks), saturation of leucine incorporation rates occurred at a lower concentration of 1.25 µM.

View larger version (21K): [in this window] [in a new window]   FIG. 1. Kinetics of [3H]leucine uptake and incorporation into protein for microbial communities associated with decaying T. angustifolia litter after 2 and 8 weeks of submergence. The line fittings illustrated were calculated by using nonlinear curve fitting to Michaelis-Menten-type kinetics. Symbols indicate the mean ± the standard error (SE) (n = 3).

View larger version (21K): [in this window] [in a new window]   FIG. 3. Kinetics of [3H]leucine uptake and incorporation into protein for microbial communities associated with decaying P. australis litter after 2 and 8 weeks of submergence. The line fittings illustrated were calculated by using nonlinear curve fitting to Michaelis-Menten-type kinetics. Symbols indicate the mean ± the SE (n = 3).

View larger version (21K): [in this window] [in a new window]   FIG. 2. Kinetics of [3H]leucine uptake and incorporation into protein for microbial communities associated with decaying S. validus litter after 2 and 8 weeks of submergence. The line fittings illustrated were calculated by using nonlinear curve fitting to Michaelis-Menten-type kinetics. Symbols indicate the mean ± the SE (n = 3).

Leucine incorporation: linearity of incorporation.
Rates of leucine incorporation displayed linearity with time up to an incubation period of 60 min for T. angustifolia and S. validus and up to 90 min for P. australis (Fig. 4). At longer incubation times (i.e., >60 min.), the rates of leucine incorporation leveled off in both T. angustifolia and S. validus.

View larger version (9K): [in this window] [in a new window]   FIG. 4. Incorporation of [3H]leucine into protein with increasing incubation times for microbial communities associated with decaying T. angustifolia, S. validus, and P. australis litter. Symbols indicate the mean ± SE (n = 3).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study revealed a biphasic response in rates of [³H]leucine incorporation into bacterial communities associated with submerged decaying wetland plant detritus. This biphasic response in the rates of leucine incorporation was relatively consistent among all plant litter types and incubation times examined, suggesting that the final concentration of leucine required to initially saturate bacterial biomass (protein) production may not change appreciably during bacterial community development on decaying litter of different wetland plant species.

These results differ markedly from previous [³H]leucine incorporation studies that reported only a uniphasic saturation response in rates of leucine incorporation (2, 26, 27). Studies by Thomaz and Wetzel (27) and Suberkropp and Weyers (26) reported a uniphasic relationship of [³H]leucine incorporation for bacterial communities associated with submerged decaying leaf litter of the freshwater emergent macrophyte, Juncus effusus, and the deciduous tree, Liriodendron tulipifera, with saturation of leucine incorporation rates occurring at a final leucine concentration of 400 nM. Similarly, Buesing and Gessner (2) also reported a uniphasic relationship of [³H]leucine incorporation into protein for bacterial communities associated with decaying leaf litter of the common reed, P. australis. However, in contrast to earlier [³H]leucine incorporation studies (26, 27), they observed that a leucine concentration of 50 µM was required to reach saturation-level uptake.

Many of the observed differences in [³H]leucine incorporation maxima between the present study and previous investigations (2, 26, 27) may be the result of large differences in the number and range of leucine concentrations examined. Early studies by Thomaz and Wetzel (27) examined [³H]leucine uptake kinetics using six leucine concentrations ranging between 10 and 800 nM. Likewise, Suberkropp and Weyers (26) examined [³H]leucine uptake kinetics using only five leucine concentrations ranging between 50 and 800 nM. Hence, the range in leucine concentrations tested by both Thomaz and Wetzel (27) and Suberkropp and Weyers (26) was not sufficiently broad enough to detect any additional saturation plateaus in [³H]leucine uptake, which could potentially occur at high (>800 nM) leucine concentrations.

In contrast to Thomaz and Wetzel (27) and Suberkropp and Weyers (26), Buesing and Gessner (2) examined six widely spaced leucine concentrations ranging between 10 nM and 100 µM. The inclusion of higher leucine concentrations in their study resulted in an observed saturation uptake of leucine at a substantially higher final leucine concentration (50 µM). However, the limited number of leucine concentrations tested within their study, especially at the lower concentrations, may have resulted in a saturation curve with insufficient resolution to observe any potential initial saturation of leucine uptake at lower concentrations.

In the present study, we examined rates of leucine incorporation using nine leucine concentrations ranging between 10 nM to 50 µM. We included additional leucine concentrations in order to provide increased resolution of leucine uptake dynamics within this large concentration range, particularly at low concentrations. When nonlinear regression analyses included all leucine uptake data for each plant species, saturation of rates of leucine incorporation into protein appeared to exhibit single uniphasic curves, with saturation of leucine incorporation occurring at final leucine concentrations of >10 µM (see Fig. 1 to 3). These results were consistent with Buesing and Gessner (2), suggesting that saturation of leucine uptake into protein may occur at much higher leucine concentrations (10 µM) than previously reported. However, when nonlinear regression analyses were restricted to a lower leucine concentration range (i.e., 5 concentrations between 10 nM and 2.5 µM), we also observed a saturation plateau in rates of leucine incorporation, indicating that natural microbial communities associated with decaying wetland plant litter may exhibit kinetic variability that yields a biphasic mode of [³H]leucine uptake.

Previous studies examining substrate uptake kinetics of natural microbial communities have reported bi- and multiphasic modes in substrate uptake (see, for example, references 8 and 30). Unanue et al. (30) examined the kinetics of bacterial hydrolytic ectoenzymatic activity and the uptake of monomeric compounds in natural bacterioplankton communities from the Mediterranean Sea. Bacterial uptake rates were examined over a wide range of substrate concentrations (0.2 nM to 3 µM) using both a mixture of radiolabeled amino acids and glucose. The results of that study revealed that marine bacterioplankton exhibited biphasic response in the uptake of glucose and amino acids, with high-affinity transport systems responsible for uptake at low substrate concentrations (K_m = 1.4 to 42 nM) and low-affinity transport systems responsible for uptake at high substrate concentrations (K_m = 0.1 to 1.3 µM). The authors postulated that the existence of these two uptake phases could be due to differing physiologic attributes within these mixed bacterial communities (e.g., high- versus low-affinity-adapted bacteria). Similar biphasic leucine uptake patterns have also been reported by Fischer and Pusch (8) for bacterial communities in freshwater stream sediments.

Differences among studies in extraction protocols for recovery of radiolabeled protein from samples may be an additional factor in the observed differences in leucine incorporation maxima between the present study and previous investigations (26, 27). Early studies by Thomaz and Wetzel (27) and Suberkropp and Weyers (26) used a modified protein extraction technique that included an initial ultrasonication step prior to TCA protein precipitation and filtration. This ultrasonication step was assumed to effectively remove attached bacteria and corresponding radiolabeled bacterial protein from the decaying litter sample. However, Buesing and Gessner (2) reported that the inclusion of an alkaline extraction (i.e., NaOH and SDS) step after ultrasonication and washing of samples significantly increased the recovery of radiolabeled protein. This finding suggest that earlier protocols used by Thomaz and Wetzel (27) and Suberkropp and Weyers (26) for extracting labeled protein from litter substrates may not accurately reflect the true rates of [³H]leucine incorporation into bacterial assemblages colonizing plant material. As a consequence, the saturation uptake kinetics of [³H]leucine incorporation reported in these studies may be an artifact reflective to the underlying extraction procedure used.

In the present study, we used an alkaline extraction method coupled with microdialysis (500 MWCO) of the final extract, instead of ultrasonication, to facilitate the removal of unincorporated radiolabel while retaining larger radiolabeled protein. Microdialysis increased the signal-to-noise ratio (i.e., signal of sample to killed control) by 20% for samples from Typha and Schoenoplectus but not for Phragmites (data not shown). However, despite microdialysis, high background levels were still observed in killed control samples, indicating the continued absorption of free [³H]leucine to leached dissolved organic compounds following alkaline extraction (see Fig. 3, step 7 in reference 2). We observed a sharp decline in signal-to-noise ratio with increasing leucine concentrations, with high final concentrations of leucine (e.g., >20 µM) having a signal-to-noise ratio of 4. Furthermore, considerable variability in leucine incorporation rates were observed among replicates at higher final leucine concentrations, probably due to a lower specific activity of leucine used within these samples and possibly also the lower signal-to-noise ratio experienced. Hence, these factors may have compromised our accurate estimation of the rates of leucine incorporation at these higher concentrations. Similar findings were also observed by Buesing and Gessner (2), who also reported high background levels (signal/noise ratio of 4) and substantial variability among sample replicates (decaying litter) at high final leucine concentrations (see Fig. 1C in reference 2).

In the present study, the rates of leucine incorporation into protein were linear for up to 90 min for P. australis and for up to 60 min for T. angustifolia and S. validus, indicating that short-term incubations at 2.5 µM leucine did not stimulate a significant increase in bacterial protein synthesis. However, we observed a departure from the linear rates of incorporation for T. angustifolia and S. validus after 90 min, suggesting that bacterial communities associated with the litter of these plant species may have depleted added leucine or shifted their metabolic uptake of leucine during this longer incubation period. These findings are consistent with previous studies (2, 26, 27) that found that leucine uptake was linear for up to 60 min, suggesting that a 30-min incubation time for leucine uptake is satisfactory over a wide range of conditions.

The presence of a biphasic saturation pattern in the rates of [³H]leucine incorporation associated with decaying plant litter may indicate potential [³H]leucine uptake and incorporation by other nonbacterial heterotrophs, particularly when high final leucine concentrations are used. Buesing and Gessner (2), using prokaryotic and eukaryotic inhibitors, speculated that the incorporation of radiolabeled [³H]leucine was primarily due to bacterial uptake and metabolism, even at leucine concentrations of 50 µM. The addition of eukaryotic inhibitors (cycloheximide and colchicines) to litter samples had no effect on the rates of leucine incorporation at 20 and 400 nM but caused a 28% reduction at 50 µM. The authors surmised that this suppression in the rates of leucine incorporation at 50 µM may have resulted from the high variability observed among replicates instead of eukaryotic inhibition, since these results were counter to additional experiments that found total suppression of leucine incorporation when bacterial inhibitors (streptomycin and chloramphenicol) were added to incubation samples. However, note that similar eukaryotic inhibitor studies by Suberkropp and Weyers (26) observed a 27% reduction in rates of leucine incorporation at a final leucine concentration of 400 nM.

Previous physiologic studies with pure cultures indicate that fungal organisms are capable of leucine uptake at high micromolar concentrations (13, 22). However, as Buesing and Gessner (2) stated, the real question is whether fungi (i.e., mycelial) successfully compete with bacteria for exogenous leucine that is added to radiolabeled incubation assays. Using previous uptake data for high-affinity leucine transport systems of Saccharomyces cerevisiae and Neurospora crassa (13), Buesing and Gessner (2) estimated that bacteria associated with decaying P. australis would take up leucine between 19 and 65 times faster than fungi at exogenous leucine concentrations of 50 µM. Consequently, fungal assemblages colonizing plant detritus could potentially contribute equally to total leucine incorporation if their metabolically active biomass were 19 to 65 times that of bacteria.

Using similar calculations, we estimated that filamentous fungi associated with decaying wetland plant litter in the present study could also contribute equally to total leucine incorporation if their metabolically active biomass were 5 to 71 times that of bacteria, depending on litter type and leucine concentration. However, like Buesing and Gessner (2), this estimate is based largely on the assumption that the leucine uptake rates of filamentous fungal organisms colonizing decaying plant litter exhibit similar uptake rates as reported for N. crassa (i.e., in reference 13 a high-affinity leucine transport system with a V_max of 0.1 nmol mg^–1 min^–1 [conidia only] and a K_t of 4.4 µM was reported, but see also reference 21, wherein a V_max of 0.44 nmol mg^–1 min^–1 in mycelia was determined). In addition, the underlying comparison of potential fungal uptake rates to that of bacteria in the present study and Buesing and Gessner (2) also assumes that the maximum rates of [³H]leucine incorporation (V_max) were entirely due to litter-associated bacterial communities.

In the present study, fungal biomass associated with decaying plant litter was between 5 and 135 times that of the bacterial biomass. The presence of substantial fungal biomass associated with decaying plant litter suggests that these eukaryotic osmotrophs might contribute to total leucine incorporation, particularly at micromolar leucine concentrations where uptake by metabolically active fungi is likely (13, 22). This uptake and incorporation of radiolabeled [³H]leucine by litter-associated fungal organisms could compromise the accurate estimation of bacterial secondary production, particularly if high final concentrations of leucine are used in the [³H]leucine radioassay and if the corresponding rates of uptake are assumed to be entirely due to bacteria. For example, if we assume that the second saturation plateau represents the true final concentration of leucine (50 µM) that should be used for quantifying rates of bacterial [³H]leucine incorporation, then bacterial communities associated with decaying T. angustifolia in the present study would have an estimated carbon production rate of 22 ± 8 mgC gC^–1 day^–1 (conversion factor: 1.44 g bacterial C/mmol leucine incorporated [see reference 4]) and a corresponding turnover time of 0.05 ± 0.01 day^–1 (Table 5). In contrast, if we assume that initial saturation of the leucine incorporation rates occurs at the first saturation plateau (2.5 µM), then the estimate of bacterial carbon production would be 6-fold less (3.6 ± 1.7 mgC gC^–1 day^–1), with a corresponding turnover time of 0.33 ± 0.18 day^–1 (Table 5). These divergent production estimates illustrate the continued uncertainty of the leucine method as a tool for measuring bacterial secondary production on decaying plant litter and may potentially explain the wide-ranging estimates of bacterial production that have been reported in the literature for decaying wetland plant litter (see, for example, references 3 and 19). Future studies using pure and mixed cultures of bacteria and fungi colonizing sterile litter substrata may help to clarify the involvement of fungal organisms in leucine uptake and further refine the final leucine concentrations that are appropriate for quantifying rates of bacterial production in mixed bacterial-fungal communities.

	ACKNOWLEDGMENTS

This research was supported by grants from the National Science Foundation (DEB-0315686 and DBI-0420965). Partial support was also provided by the Department of Biology and the Graduate School of Eastern Michigan University.

	FOOTNOTES

 
* 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.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Buesing, N., and M. O. Gessner. 2002. Comparison of detachment procedures for direct counts of bacteria associated with sediment particles, plant litter, and epiphytic biofilms. Aquat. Microb. Ecol. 27:29-36.
2. Buesing, N., and M. O. Gessner. 2003. Incorporation of radiolabelled leucine into protein to estimate bacterial production in plant litter, sediment, epiphytic biofilms, and water samples. Microb. Ecol. 45:291-301.[CrossRef][Medline]
3. Buesing, N., and M. O. Gessner. 2006. Benthic bacterial and fungal productivity and carbon turnover in a freshwater marsh. Appl. Environ. Microbiol. 72:596-605.[Abstract/Free Full Text]
4. Buesing, N., and J. Marxsen. 2005. Theoretical and empirical conversion factors for determining bacterial production in freshwater sediments via leucine incorporation. Limnol. Oceanogr. Methods 3:101-107.
5. Cole, J. J., S. Findlay, and M. L. Pace. 1988. Bacterial production in freshwater and saltwater ecosystems: a cross-system overview. Mar. Ecol. 43:1-10.
6. Dvorák, J., and G. Imhof. 1998. The role of animals and animal communities in wetlands, p. 211-318. In D. F. Westlake, J. Kvet, and A. Szczepanski (ed.), The production ecology of wetlands. Cambridge University Press, Cambridge, United Kingdom.
7. Findlay, S. E. G., S. Dye, and K. A. Kuehn. 2002. Microbial growth and nitrogen retention in litter of Phragmites australis compared to Typha angustifolia. Wetlands 22:616-625.[CrossRef]
8. Fischer, H., and M. Pusch. 1999. Use of the [¹⁴C]leucine incorporation technique to measure bacterial production in river sediments and the epiphyton. Appl. Environ. Microbiol. 65:4411-4418.[Abstract/Free Full Text]
9. Fuhrman, J. A., and F. Azam. 1980. Thymidine incorporation into DNA as a measure of heterotrophic bacterioplankton production in marine surface waters: evaluation and field results. Appl. Environ. Microbiol. 39:1085-1095.[Abstract/Free Full Text]
10. Furtado, A. L. S., and P. Casper. 2000. Different methods for extracting bacteria from freshwater sediment and a simple method to measure bacterial production in sediment samples. J. Microbiol. Methods 41:249-257.[CrossRef][Medline]
11. Gessner, M. O., and S. Y. Newell. 2002. Biomass, growth rate, and production of filamentous fungi in plant litter, p. 390-408. In C. J. Hurst, R. L. Crawford, G. R. Knudsen, M. J. McInerney, and L. D. Stetzenbach (ed.), Manual of environmental microbiology, 2nd ed. ASM Press, Washington, D.C.
12. Gulis, V., and K. Suberkropp. 2003. Leaf litter decomposition and microbial activity in nutrient-enriched and unaltered reaches of a headwater stream. Freshwater Biol. 48:123-134.[CrossRef]
13. Horák, J. 1986. Amino acid transport in eukaryotic microorganisms. Biochim. Biophys. Acta 864:223-256.[Medline]
14. Jorgensen, N. O. G. 1992. Incorporation of [³H]leucine and [³H]valine into protein of freshwater bacteria: field applications. Appl. Environ. Microbiol. 58:3647-3653.[Abstract/Free Full Text]
15. Kirchman, D., E. K'Nees, and R. Hodson. 1985. Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems. Appl. Environ. Microbiol. 49:599-607.[Abstract/Free Full Text]
16. Kirschner, A. K. T., and B. Velimirov. 1999. Benthic bacterial secondary production measured via simultaneous ³H-thymidine and ¹⁴C-leucine incorporation, and its implication for the carbon cycle of a shallow macrophyte-dominated backwater system. Limnol. Oceanogr. 44:1871-1881.
17. Komínková, D., K. A. Kuehn, N. Buesing, D. Steiner, and M. O. Gessner. 2000. Microbial biomass, growth, and respiration associated with submerged litter of Phragmites australis decomposing in a littoral reed stand of a large lake. Aquat. Microb. Ecol. 22:271-282.
18. Kuehn, K. A., and K. Suberkropp. 1998. Decomposition of standing litter of the freshwater macrophyte Juncus effusus. Freshwater Biol. 40:717-727.[CrossRef]
19. Kuehn, K. A., M. J. Lemke, K. Suberkropp, and R. G. Wetzel. 2000. Microbial biomass and production associated with decaying leaf litter of the emergent macrophyte Juncus effusus. Limnol. Oceanogr. 45:862-870.
20. Kuehn, K. A., D. Steiner, and M. O. Gessner. 2004. Diel mineralization patterns of standing dead litter: implications for CO₂ flux from temperate wetlands. Ecology 89:2504-2518.
21. Noble, R. T., and J. A. Fuhrman. 1998. Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria. Aquat. Microb. Ecol. 14:113-118.
22. Pall, M. L. 1969. Amino acid transport in Neurospora crassa. I. Properties of two amino acid transport systems. Biochim. Biophys. Acta 173:113-127.[Medline]
23. Petit, M., P. Servais, and P. Lavandier. 1999. Bacterial production measured by leucine and thymidine incorporation rates in French Lakes. Freshwater Biol. 42:513-524.[CrossRef]
24. Simon, M., and F. Azam. 1989. Protein content and protein synthesis rates of planktonic marine bacteria. Mar. Ecol. Prog. Ser. 51:201-213.
25. Simon, M., H. P. Grossart, B. Schweitzer, and H. Ploug. 2002. Microbial ecology of organic aggregates in aquatic ecosystems. Aquat. Microb. Ecol. 28:175-211.
26. Suberkropp, K., and H. Weyers. 1996. Application of fungal and bacterial production methodologies to decomposing leaves in streams. Appl. Environ. Microbiol. 62:1610-1615.[Abstract]
27. Thomaz, S. M., and R. G. Wetzel. 1995. [³H]leucine incorporation methodology to estimate epiphytic bacterial biomass production. Microb. Ecol. 29:63-70.
28. Thomaz, S. M., and F. A. Esteves. 1997. Secondary productivity (³H-leucine and ³H-thymidine incorporation), abundance, and biomass of the epiphytic bacteria attached to detritus of Typha domingensis in a tropical coastal lagoon. Hydrobiologia 357:17-26.[CrossRef]
29. Tuominen, L. 1995. Comparison of leucine uptake methods and a thymidine incorporation method for measuring bacterial activity in sediment. J. Microbiol. Methods 24:125-134.[CrossRef]
30. Unanue, M., B. Ayo, M. Agis, D. Slezak, G. J. Herndl, and J. Iriberri. 1999. Ectoenzymatic activity and uptake of monomers in marine bacterioplankton described by a biphasic kinetic model. Microb. Ecol. 37:36-48.[CrossRef][Medline]
31. Wetzel, R. G. 1990. Land-water interfaces: metabolic and limnological regulators. Int. Ver. Theor. Angew. Limnol. Verh. 24:6-24.
32. Wetzel, R. G., and G. E. Likens. 1991. Limnological analyses, 2nd ed. Springer Verlag, New York, N.Y.
33. Wetzel, R. G., and M. J. Howe. 1999. High production in a herbaceous perennial plant achieved by continuous growth and synchronized population dynamics. Aquat. Bot. 64:111-129.[CrossRef]
34. Weyers, H., and K. Suberkropp. 1996. Fungal and bacterial production during the breakdown of yellow poplar leaves in 2 streams. J. N. Am. Benthol. Soc. 15:408-420.[CrossRef]
35. Windham, L. 2001. Comparison of biomass production and decomposition between Phragmites australis (common reed) and Spartina patens (salt hay grass) in brackish tidal marshes of New Jersey, USA. Wetlands 21:179-188.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Gillies, J. E. Articles by Neely, R. K. Search for Related Content PubMed PubMed Citation Articles by Gillies, J. E. Articles by Neely, R. K. Agricola Articles by Gillies, J. E. Articles by Neely, R. K.