Use of the [14C]Leucine Incorporation Technique To Measure Bacterial Production in River Sediments and the Epiphyton

Bacterial production is a key parameter for the understanding of carbon cycling in aquatic ecosystems, yet it remains difficult to measure in many aquatic habitats. We therefore tested the applicability of the [¹⁴C]leucine incorporation technique for the measurement of bulk bacterial production in various habitats of a lowland river ecosystem. To evaluate the method, we determined (i) extraction efficiencies of bacterial protein from the sediments, (ii) substrate saturation of leucine in sediments, the biofilms on aquatic plants (epiphyton), and the pelagic zone, (iii) bacterial activities at different leucine concentrations, (iv) specificity of leucine uptake by bacteria, and (v) the effect of the incubation technique (perfused-core incubation versus slurry incubation) on leucine incorporation into protein. Bacterial protein was best extracted from sediments and precipitated by hot trichloroacetic acid treatment following ultrasonication. For epiphyton, an alkaline-extraction procedure was most efficient. Leucine incorporation saturation occurred at 1 μM in epiphyton and 100 nM in the pelagic zone. Saturation curves in sediments were difficult to model but showed the first level of leucine saturation at 50 μM. Increased uptake at higher leucine concentrations could be partly attributed to eukaryotes. Addition of micromolar concentrations of leucine did not enhance bacterial electron transport activity or DNA replication activity. Similar rates of leucine incorporation into protein calculated for whole sediment cores were observed after slurry and perfused-core incubations, but the rates exhibited strong vertical gradients after the core incubation. We conclude that the leucine incorporation method can measure bacterial production in a wide range of aquatic habitats, including fluvial sediments, if substrate saturation and isotope dilution are determined.     

Comparison of bacterial production in sediments, epiphyton and the pelagic zone of a lowland river

1. The microbial metabolism of organic matter in rivers has received little study compared with that of small streams. Therefore, we investigated the rate and location of bacterial production in a sixth‐order lowland river (Spree, Germany). To estimate the contribution of various habitats (sediments, epiphyton, and the pelagic zone) to total bacterial production, we quantified the contribution of these habitats to areal production by bacteria. 2. Large areas of the river bottom were characterized by loose and shifting sands of relatively homogenous particle size distribution. Aquatic macrophytes grew on 40% of the river bottom. Leaf areas of 2.8 m² m^?2 river bottom were found in a 6.6 km river stretch. 3. The epiphyton supported a bacterial production of 5–58 ng C cm^?2 h^?1. Bacterial production in the pelagic zone was 0.9–3.9 μg C L^?1 h^?1, and abundance was 4.0–7.8 × 10⁹ cells L^?1. Bacterial production in the uppermost 2 cm of sediments ranged from 1 to 8 μg C cm^?3 h^?1, and abundance from 0.84 to 6.7 × 10⁹ cells cm^?3. Bacteria were larger and more active in sediments than in the pelagic zone. 4. In spite of relatively low macrophyte abundance, areal production by bacteria in the pelagic zone was only slightly higher than in the epiphyton. Bacterial biomass in the uppermost 2 cm of sediments exceeded pelagic biomass by factors of 6–22, and sedimentary bacterial production was 17–35 times higher than in the overlying water column. 5. On a square meter basis, total bacterial production in the Spree was clearly higher than primary productivity. Thus, the lowland river Spree is a heterotrophic system with benthic processes dominating. Therefore, sedimentary and epiphytic bacterial productivity form important components of ecosystem carbon metabolism in rivers and shallow lakes. 6. The sediments are focal sites of microbial degradation of organic carbon in a sand‐bottomed lowland river. The presence of a lowland river section within a river continuum probably greatly changes the geochemical fluxes within the river network. This implies that current concepts of longitudinal biogeochemical relationships within river systems have to be revised.     

The Leucine Incorporation Method Estimates Bacterial Growth Equally Well in Both Oxic and Anoxic Lake Waters

Bacterial biomass production is often estimated from incorporation of radioactively labeled leucine into protein, in both oxic and anoxic waters and sediments. However, the validity of the method in anoxic environments has so far not been tested. We compared the leucine incorporation of bacterial assemblages growing in oxic and anoxic waters from three lakes differing in nutrient and humic contents. The method was modified to avoid O₂ contamination by performing the incubation in syringes. Isotope saturation levels in oxic and anoxic waters were determined, and leucine incorporation rates were compared to microscopically observed bacterial growth. Finally, we evaluated the effects of O₂ contamination during incubation with leucine, as well as the potential effects of a headspace in the incubation vessel. Isotope saturation occurred at a leucine concentration of above about 50 nM in both oxic and anoxic waters from all three lakes. Leucine incorporation rates were linearly correlated to observed growth, and there was no significant difference between oxic and anoxic conditions. O₂ contamination of anoxic water during 1-h incubations with leucine had no detectable impact on the incorporation rate, while a headspace in the incubation vessel caused leucine incorporation to increase in both anoxic and O₂-contaminated samples. The results indicate that the leucine incorporation method relates equally to bacterial growth rates under oxic and anoxic conditions and that incubation should be performed without a headspace.     

The leucine incorporation method estimates bacterial growth equally well in both oxic and anoxic lake waters.

Bacterial biomass production is often estimated from incorporation of radioactively labeled leucine into protein, in both oxic and anoxic waters and sediments. However, the validity of the method in anoxic environments has so far not been tested. We compared the leucine incorporation of bacterial assemblages growing in oxic and anoxic waters from three lakes differing in nutrient and humic contents. The method was modified to avoid O(2) contamination by performing the incubation in syringes. Isotope saturation levels in oxic and anoxic waters were determined, and leucine incorporation rates were compared to microscopically observed bacterial growth. Finally, we evaluated the effects of O(2) contamination during incubation with leucine, as well as the potential effects of a headspace in the incubation vessel. Isotope saturation occurred at a leucine concentration of above about 50 nM in both oxic and anoxic waters from all three lakes. Leucine incorporation rates were linearly correlated to observed growth, and there was no significant difference between oxic and anoxic conditions. O(2) contamination of anoxic water during 1-h incubations with leucine had no detectable impact on the incorporation rate, while a headspace in the incubation vessel caused leucine incorporation to increase in both anoxic and O(2)-contaminated samples. The results indicate that the leucine incorporation method relates equally to bacterial growth rates under oxic and anoxic conditions and that incubation should be performed without a headspace.     

3H]Leucine incorporation method as a tool to measure secondary production by periphytic bacteria associated to the roots of floating aquatic macrophyte

The present study assessed the application of [(3)H]Leucine incorporation into protein by periphytic bacteria associated with the roots of the floating aquatic macrophyte Eichornia crassipes. Basic assumptions underlying the method, such as linearity of leucine incorporation, saturation level of incorporation rates, incorporation into other macromolecules, specificity of incorporation for bacterial assemblages and [(3)H]Leucine degradation during samples storage were tested, and two procedures for extracting the incorporated leucine were compared. Both methods gave the same results, however, the hot TCA extraction method was less time consuming than the alkaline extraction method. Incorporation of [(3)H]Leucine was linear for up to 40 min. Saturation concentration of [(3)H]Leucine incorporation into protein was 1500 nM. An experiment with prokaryotic and eukaryotic inhibitors showed no significant [(3)H]Leucine incorporation into eukaryotes even in high leucine concentrations. No significant amounts of radiolabel were incorporated into other macromolecules. The maximum time of sample storage after the incubation is 15 days. The leucine incorporation method can be a reliable tool to measure bacterial production in the periphyton root-associated bacteria.     

Measurement of bacterial production in stream-bed sediments via leucine incorporation

Abstract: The leucine incorporation technique was evaluated and optimised for measuring bacterial production in stream-bed sediments. The original procedure was modified in order to obtain reliable production estimates in this habitat. This included the use of higher leucine concentrations (50 μmol l⁻¹) for obtaining substrate saturation, and an enhanced protein extraction procedure after sample fixation. The leucine method was combined with a perfused core technique. Water containing ¹⁴C-labelled leucine was perfused up through sediment cores, enabling the measurement of bacterial production in an experimental situation resembling natural conditions in the stream bed. Bacterial production in the Breitenbach, a small upland stream in Central Germany, showed a high degree of spatial variability in the sandy stream bed. It was related closely to sediment organic matter content, whereas varying perfusion rates had less influence. Annual bacterial production was estimated at about 200 g C m⁻², demonstrating the potential for bacteria to act as a food resource for benthic fauna in this stream.     

Application of Fungal and Bacterial Production Methodologies to Decomposing Leaves in Streams

As leaves enter woodland streams, they are colonized by both fungi and bacteria. To determine the contribution of each of these microbial groups to the decomposition process, comparisons of fungal and bacterial production are needed. Recently, a new method for estimating fungal production based on rates of [(sup14)C]acetate incorporation into ergosterol was described. Bacterial production in environmental samples has been determined from rates of [(sup3)H]leucine incorporation into protein. In this study, we evaluated conditions necessary to use these methods for estimating fungal and bacterial production associated with leaves decomposing in a stream. During incubation of leaf disks with radiolabeled substrates, aeration increased rates of fungal incorporation but decreased bacterial production. Incorporation of both radiolabeled substrates by microorganisms associated with leaf litter was linear over the time periods examined (2 h for bacteria and 4 h for fungi). Incorporation of radiolabeled substrates present at different concentrations indicated that 400 nM leucine and 5 mM acetate maximized uptake for bacteria and fungi, respectively. Growth rates and rates of acetate incorporation into ergosterol followed similar patterns when fungi were grown on leaf disks in the laboratory. Three species of stream fungi exhibited similar ratios of rates of biomass increase to rates of acetate incorporation into ergosterol, with a mean of 19.3 (mu)g of biomass per nmol of acetate incorporated. Both bacterial and fungal production increased exponentially with increasing temperature. In the stream that we examined, fungal carbon production was 11 to 26 times greater than bacterial carbon production on leaves colonized for 21 days.     

Incorporation of [h]leucine and [h]valine into protein of freshwater bacteria: uptake kinetics and intracellular isotope dilution

Incorporation of [H]leucine and [H]valine into proteins of freshwater bacteria was studied in two eutrophic lakes. Incorporation of both amino acids had a saturation level of about 50 nM external concentration. Only a fraction of the two amino acids taken up was used in protein synthesis. At 100 nM, the bacteria respired 91 and 78% of leucine and valine taken up, respectively. Respiration of H and C isotopes of leucine gave similar results. Most of the nonrespired leucine was recovered in bacterial proteins, while only up to one-half of the nonrespired valine occurred in proteins. In intracellular pools of the bacteria, [H]leucine reached an isotope saturation of 88 to 100% at concentrations of >40 nM. For [H]valine, an isotope equilibrium of about 90% was obtained at concentrations of >80 nM. Within an incubation period of typically 1 h, tritiated leucine and valine incorporated into proteins of the bacteria reached an isotope saturation of 2 to 6%. In a 99-h batch experiment, bacterial protein synthesis calculated from incorporation of leucine and valine corresponded to 31 and 51% (10 nM) and 89 and 97% (100 nM), respectively, of the chemically determined protein production. Measured conversion factors of 100 nM leucine and valine were 6.4 x 10 and 6.6 x 10 cells per mol, respectively, and fell within the expected theoretical values. The present study demonstrates that incorporation of both valine and leucine produces realistic measurements of protein synthesis in freshwater bacteria and that the incorporation can be used as a measure of bacterial production.     

Incorporation of [3H]Leucine and [3H]Valine into Protein of Freshwater Bacteria: Field Applications

Incorporation of leucine and valine into proteins of freshwater bacteria as a measure of bacterial production was tested in two eutrophic Danish lakes and was related to bacterial production measured by thymidine incorporation. In a depth profile (0 to 8 m) in Frederiksborg Castle Lake, incorporation of 100 nM leucine and valine gave similar rates of protein production. In terms of carbon, this production was about 50% lower than incorporation of 10 nM thymidine. In another depth profile in the same lake, incorporations of 10 nM valine and 100 nM leucine were identical, but differed from incorporations of 10 nM leucine and 100 nM valine. Bacterial carbon production calculated from incorporations of 10 nM thymidine and 10 nM leucine was similar, whereas 10 nM valine and 100 nM leucine and valine indicated an up to 2.4-fold-higher rate of carbon production. In a diel study in Lake Bagsvaerd, incorporation of 100 nM leucine and valine indicated a similar protein production, but the calculated carbon production was about 1.9-fold higher than the production based on uptake of 10 nM thymidine. Different diel changes in incorporation of the two amino acids and in incorporation of thymidine were observed. In both lakes, concentrations of naturally occurring leucine and valine were <5 nM in most samples. This means that the specific activity of a ³H isotope added at a concentration of 100 nM usually was diluted a maximum of 5%. Net assimilation of natural free amino acids in the lakes sustained 8 to 69% of the net bacterial carbon requirement, estimated from incorporation of leucine, valine, or thymidine. The present results indicate that incorporation of leucine and valine permits realistic measurements of bacterial production in freshwater environments.     

Incorporation of [h]leucine and [h]valine into protein of freshwater bacteria: field applications

Incorporation of leucine and valine into proteins of freshwater bacteria as a measure of bacterial production was tested in two eutrophic Danish lakes and was related to bacterial production measured by thymidine incorporation. In a depth profile (0 to 8 m) in Frederiksborg Castle Lake, incorporation of 100 nM leucine and valine gave similar rates of protein production. In terms of carbon, this production was about 50% lower than incorporation of 10 nM thymidine. In another depth profile in the same lake, incorporations of 10 nM valine and 100 nM leucine were identical, but differed from incorporations of 10 nM leucine and 100 nM valine. Bacterial carbon production calculated from incorporations of 10 nM thymidine and 10 nM leucine was similar, whereas 10 nM valine and 100 nM leucine and valine indicated an up to 2.4-fold-higher rate of carbon production. In a diel study in Lake Bagsvaerd, incorporation of 100 nM leucine and valine indicated a similar protein production, but the calculated carbon production was about 1.9-fold higher than the production based on uptake of 10 nM thymidine. Different diel changes in incorporation of the two amino acids and in incorporation of thymidine were observed. In both lakes, concentrations of naturally occurring leucine and valine were <5 nM in most samples. This means that the specific activity of a H isotope added at a concentration of 100 nM usually was diluted a maximum of 5%. Net assimilation of natural free amino acids in the lakes sustained 8 to 69% of the net bacterial carbon requirement, estimated from incorporation of leucine, valine, or thymidine. The present results indicate that incorporation of leucine and valine permits realistic measurements of bacterial production in freshwater environments.     

Assessment of [h]thymidine incorporation into DNA as a method to determine bacterial productivity in stream bed sediments

We performed several checks on the underlying assumptions and procedures of the thymidine technique applied to stream bed sediments. Bacterial production rates were not altered when sediments were mixed to form a slurry. Incubation temperature did affect production rates. Controls fixed and washed with formaldehyde had lower backgrounds than trichloroacetic acid controls. DNA extraction by base hydrolysis was incomplete and variable at 25 degrees C, but hydrolysis at 120 degrees C extracted 100% of the DNA, of which 84% was recovered upon precipitation. Production rates increased as thymidine concentrations were increased over 3 orders of magnitude (30 nM to 53 muM thymidine). However, over narrower concentration ranges, thymidine incorporation into DNA was independent of thymidine concentration. Elevated exogenous thymidine concentrations did not eliminate de novo synthesis. Transport of thymidine into bacterial cells occurred at least 5 to 20 times faster than incorporation of label into DNA. We found good agreement between production rates of bacterial cultures based upon increases in cell numbers and estimates based upon thymidine incorporation and amount of DNA per cell. Those comparisons emphasized the importance of isotopic dilution measurements and validated the use of the reciprocal plot technique for estimating isotopic dilution. Nevertheless, the thymidine technique cannot be considered a routine assay and the inability to measure the cellular DNA content in benthic communities restricts the accuracy of the method in those habitats.     

Depth Distribution of Bacterial Production in a Stratified Lake with an Anoxic Hypolimnion

The purpose of this study was to determine the depth distribution of bacterial biomass and production in a stratified lake and to test techniques to measure bacterial production in anaerobic waters. Bacterial abundance and incorporation of both [³H]thymidine and [³H]leucine into protein were highest in the metalimnion, at the depth at which oxygen first became unmeasurable. In contrast, [³H]thymidine incorporation into DNA was highest in the epilimnion. The ratios of incorporation into DNA/protein averaged 2.2, 0.49, and 0.95 for the epilimnion, metalimnion, and hypolimnion, respectively. Low incorporation into DNA was not due to artifacts associated with the DNA isolation procedure. Recovery of added [³H]DNA was about 90% in waters in which the portion of [³H]thymidine incorporation into DNA was about 40%. At least some obligate anaerobic bacteria were capable of assimilating thymidine since aeration of anaerobic hypolimnion waters substantially inhibited thymidine incorporation. The depth profile of bacterial production estimated from total thymidine and leucine incorporation and the frequency of dividing cells were all similar, with maximal rates in the metalimnion. However, estimates of bacterial production based on frequency of dividing cells and leucine incorporation were usually significantly higher than estimates based on thymidine incorporation (using conversion factors from the literature), especially in anaerobic hypolimnion waters. These data indicate that the thymidine approach must be examined carefully if it is to be applied to aquatic systems with low oxygen concentrations. Our results also indicate that the interface between the aerobic epilimnion and anaerobic hypolimnion is the site of intense bacterial mineralization and biomass production which deserves further study.     

Benthic carbon is inefficiently transferred in the food webs of two eutrophic shallow lakes

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Incorporation of [3H]Leucine and [3H]Valine into Protein of Freshwater Bacteria: Uptake Kinetics and Intracellular Isotope Dilution

Incorporation of [³H]leucine and [³H]valine into proteins of freshwater bacteria was studied in two eutrophic lakes. Incorporation of both amino acids had a saturation level of about 50 nM external concentration. Only a fraction of the two amino acids taken up was used in protein synthesis. At 100 nM, the bacteria respired 91 and 78% of leucine and valine taken up, respectively. Respiration of ³H and ¹⁴C isotopes of leucine gave similar results. Most of the nonrespired leucine was recovered in bacterial proteins, while only up to one-half of the nonrespired valine occurred in proteins. In intracellular pools of the bacteria, [³H]leucine reached an isotope saturation of 88 to 100% at concentrations of >40 nM. For [³H]valine, an isotope equilibrium of about 90% was obtained at concentrations of >80 nM. Within an incubation period of typically 1 h, tritiated leucine and valine incorporated into proteins of the bacteria reached an isotope saturation of 2 to 6%. In a 99-h batch experiment, bacterial protein synthesis calculated from incorporation of leucine and valine corresponded to 31 and 51% (10 nM) and 89 and 97% (100 nM), respectively, of the chemically determined protein production. Measured conversion factors of 100 nM leucine and valine were 6.4 × 10¹⁶ and 6.6 × 10¹⁶ cells per mol, respectively, and fell within the expected theoretical values. The present study demonstrates that incorporation of both valine and leucine produces realistic measurements of protein synthesis in freshwater bacteria and that the incorporation can be used as a measure of bacterial production.     

Measurement of the incorporation rates of four amino acids into proteins for estimating bacterial production

In aquatic ecosystems, [³H]thymidine incorporation into bacterial DNA and [³H]leucine incorporation into proteins are usually used to estimate bacterial production. The incorporation rates of four amino acids (leucine, tyrosine, lysine, alanine) into proteins of bacteria were measured in parallel on natural freshwater samples from the basin of the river Meuse (Belgium). Comparison of the incorporation into proteins and into the total macromolecular fraction showed that these different amino acids were incorporated at more than 90% into proteins. From incorporation measurements at four subsaturated concentrations (range, 2–77 nm), the maximum incorporation rates were determined. Strong correlations (r > 0.91 for all the calculated correlations) were found between the maximum incorporation rates of the different tested amino acids over a range of two orders of magnitude of bacterial activity. Bacterial production estimates were calculated using theoretical and experimental conversion factors. The productions calculated from the incorporation rates of the four amino acids were in good concordance, especially when the experimental conversion factors were used (slope range, 0.91–1.11, and r > 0.91). This study suggests that the incorporation of various amino acids into proteins can be used to estimate bacterial production.     

Assessment of [3H]Thymidine Incorporation into DNA as a Method To Determine Bacterial Productivity in Stream Bed Sediments

We performed several checks on the underlying assumptions and procedures of the thymidine technique applied to stream bed sediments. Bacterial production rates were not altered when sediments were mixed to form a slurry. Incubation temperature did affect production rates. Controls fixed and washed with formaldehyde had lower backgrounds than trichloroacetic acid controls. DNA extraction by base hydrolysis was incomplete and variable at 25°C, but hydrolysis at 120°C extracted 100% of the DNA, of which 84% was recovered upon precipitation. Production rates increased as thymidine concentrations were increased over 3 orders of magnitude (30 nM to 53 μM thymidine). However, over narrower concentration ranges, thymidine incorporation into DNA was independent of thymidine concentration. Elevated exogenous thymidine concentrations did not eliminate de novo synthesis. Transport of thymidine into bacterial cells occurred at least 5 to 20 times faster than incorporation of label into DNA. We found good agreement between production rates of bacterial cultures based upon increases in cell numbers and estimates based upon thymidine incorporation and amount of DNA per cell. Those comparisons emphasized the importance of isotopic dilution measurements and validated the use of the reciprocal plot technique for estimating isotopic dilution. Nevertheless, the thymidine technique cannot be considered a routine assay and the inability to measure the cellular DNA content in benthic communities restricts the accuracy of the method in those habitats.     

Contrasting ability to take up leucine and thymidine among freshwater bacterial groups: implications for bacterial production measurements

We examined the ability of different freshwater bacterial groups to take up leucine and thymidine in two lakes. Utilization of both substrates by freshwater bacteria was examined at the community level by looking at bulk incorporation rates and at the single-cell level by combining fluorescent in situ hybridization and signal amplification by catalysed reporter deposition with microautoradiography. Our results showed that leucine was taken up by 70–80% of Bacteria -positive cells, whereas only 15–43% of Bacteria -positive cells were able to take up thymidine. When a saturating substrate concentration in combination with a short incubation was used, 80–90% of Betaproteobacteria and 67–79% of Actinobacteria were positive for leucine uptake, whereas thymidine was taken up by < 10% of Betaproteobacteria and by < 1% of the R-BT subgroup that dominated this bacterial group. Bacterial abundance was a good predictor of the relative contribution of bacterial groups to leucine uptake, whereas when thymidine was used Actinobacteria represented the large majority (> 80%) of the cells taking up this substrate. Increasing the substrate concentration to 100 nM did not affect the percentage of R-BT cells taking up leucine (> 90% even at low concentrations), but moderately increased the fraction of thymidine-positive R-BT cells to a maximum of 35% of the hybridized cells. Our results show that even at very high concentrations, thymidine is not taken up by all, otherwise active, bacterial cells.     

Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems.

Leucine incorporation was examined as a method for estimating rates of protein synthesis by bacterial assemblages in natural aquatic systems. The proportion of the total bacterial population that took up leucine in three marine environments was high (greater than 50%). Most of the leucine (greater than 90%) taken up was incorporated into protein, and little (less than 20%) was degraded to other amino acids, except in two oligotrophic marine environments. In samples from these two environments, ca. 50% of the leucine incorporated had been degraded to other amino acids, which were subsequently incorporated into protein. The degree of leucine degradation appears to depend on the organic carbon supply, as the proportion of 3H-radioactivity incorporated into protein that was recovered as [3H]leucine after acid hydrolysis increased with the addition of pyruvate to oligotrophic water samples. The addition of extracellular leucine inhibited total incorporation of [14C]pyruvate (a precursor for leucine biosynthesis) into protein. Furthermore, the proportion of [14C]pyruvate incorporation into protein that was recovered as [14C]leucine decreased with the addition of extracellular leucine. These results show that the addition of extracellular leucine inhibits leucine biosynthesis by marine bacterial assemblages. The molar fraction of leucine in a wide variety of proteins is constant, indicating that changes in leucine incorporation rates reflect changes in rates of protein synthesis rather than changes in the leucine content of proteins. The results demonstrate that the incorporation rate of [3H]leucine into a hot trichloroacetic acid-insoluble cell fraction can serve as an index of protein synthesis by bacterial assemblages in aquatic systems.     

Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems

Leucine incorporation was examined as a method for estimating rates of protein synthesis by bacterial assemblages in natural aquatic systems. The proportion of the total bacterial population that took up leucine in three marine environments was high (greater than 50%). Most of the leucine (greater than 90%) taken up was incorporated into protein, and little (less than 20%) was degraded to other amino acids, except in two oligotrophic marine environments. In samples from these two environments, ca. 50% of the leucine incorporated had been degraded to other amino acids, which were subsequently incorporated into protein. The degree of leucine degradation appears to depend on the organic carbon supply, as the proportion of 3H-radioactivity incorporated into protein that was recovered as [3H]leucine after acid hydrolysis increased with the addition of pyruvate to oligotrophic water samples. The addition of extracellular leucine inhibited total incorporation of [14C]pyruvate (a precursor for leucine biosynthesis) into protein. Furthermore, the proportion of [14C]pyruvate incorporation into protein that was recovered as [14C]leucine decreased with the addition of extracellular leucine. These results show that the addition of extracellular leucine inhibits leucine biosynthesis by marine bacterial assemblages. The molar fraction of leucine in a wide variety of proteins is constant, indicating that changes in leucine incorporation rates reflect changes in rates of protein synthesis rather than changes in the leucine content of proteins. The results demonstrate that the incorporation rate of [3H]leucine into a hot trichloroacetic acid-insoluble cell fraction can serve as an index of protein synthesis by bacterial assemblages in aquatic systems.