Leucine Incorporation and Its Potential as a Measure of Protein Synthesis by Bacteria in Natural Aquatic Systemst

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1 APPLD AND NVRONMNTAL MCROBOLOGY, Mar. 1985, p /85/3599-9$2./ Copyright C 1985, Amerian Soiety for Mirobiology Vol. 49, No. 3 Leuine norporation and ts Potential as a Measure of Protein Synthesis by Bateria in Natural Aquati Systemst DAVD KRCHMAN,+* LZABTH K'NS, AND ROBRT HODSON Department of Mirobiology and nstitute of ology, University of Georgia, Athens, Georgia 362 Reeived 23 July 1984/Aepted 21 Deember 1984 Leuine inorporation was examined as a method for estimating rates of protein synthesis by baterial assemblages in natural aquati systems. The proportion of the total baterial population that took up leuine in three marine environments was high (>5%). Most of the leuine (>9%) taken up was inorporated into protein, and little (<2%) was degraded to other amino aids, exept in two oligotrophi marine environments. n samples from these two environments, a. 5% of the leuine inorporated had been degraded to other amino aids, whih were subsequently inorporated into protein. The degree of leuine degradation appears to depend on the organi arbon supply, as the proportion of 3H-radioativity inorporated into protein that was reovered as [3H]leuine after aid hydrolysis inreased with the addition of pyruvate to oligotrophi water samples. The addition of extraellular leuine inhibited total inorporation of [14C]pyruvate (a preursor for leuine biosynthesis) into protein. Furthermore, the proportion of [14C]pyruvate inorporation into protein that was reovered as [14C]leuine dereased with the addition of extraellular leuine. These results show that the addition of extraellular leuine inhibits leuine biosynthesis by marine baterial assemblages. The molar fration of leuine in a wide variety of proteins is onstant, indiating that hanges in leuine inorporation rates reflet hanges in rates of protein synthesis rather than hanges in the leuine ontent of proteins. The results demonstrate that the inorporation rate of [3H]leuine into a hot trihloroaeti aid-insoluble ell fration an serve as an index of protein synthesis by baterial assemblages in aquati systems. The rate of protein synthesis ould be a valuable parameter for evaluating the role of bateria in trophi dynamis and biogeohemial yles of aquati systems. Protein is a major maromoleule in baterial ells and onstitutes over half of the dry weight of bateria in pure ulture (14) and in mixed marine assemblages (8). Thus, protein synthesis per se must be a signifiant fration of total biomass prodution. Sine protein synthesis depends on the utilization of nitrogenous ompounds taken up from the environment, the rate of protein synthesis may be a more diret measure than estimates derived from total baterial prodution of nitrogen utilization by heterotrophi bateria in aquati systems. The rate of protein synthesis would also aount for a large fration of the rate of utilization of ompounds for energy prodution, sine 96% of the total energy expended for synthesizing maromoleules by sherihia oli, for example, is used to synthesize proteins (14). Furthermore, beause protein is a valuable food soure for heterotrophi organisms (31), rates of net baterial protein synthesis may reflet more aurately than estimates of total baterial prodution the ontribution of bateria to the energeti needs of organisms that graze on bateria. Rates of protein synthesis in total mirobial assemblages have been estimated from the inorporation of 35SO4 (4). However, there are two problems with the appliation of this tehnique to baterial assemblages, espeially in marine environments. First, phytoplankton are responsible for a large proportion of total SO4 uptake (84% in a freshwater lake [15]), and thus hanges in rates of 15 O4 inorporation ould be due to either the phytoplankton or the bateria. * Corresponding author. t Contribution no. 532 of the Marine nstitute, University of Georgia, Sapelo sland, GA t Present address: Department of Moleular Genetis and Cell Biology, The University of Chiago, Chiago, L Seond, the inorporation of 35SO4 is diffiult to measure in marine environments beause the high onentration of sulfate in seawater isotopially dilutes the added 35SO4. For these reasons we hose to examine the inorporation of leuine to omplement the existing method based on 35 o4 inorporation. Leuine inorporation is often used in laboratory studies as an index of protein synthesis (for examples, see referenes 22, 32, and 37). n its simplest form, the method onsists of adding radiolabeled leuine and measuring the aumulation of radioativity in the hot trihloroaeti aid (TCA)-insoluble ell fration olleted on membrane filters. The hot TCA extrat ontains protein, peptidoglyan, and perhaps some omplex polysaharides (1, 29). The use of leuine inorporation rates to estimate the rate of protein synthesis by baterial assemblages in aquati environments has not been examined. This paper reports several experiments on leuine uptake and biosynthesis by marine baterial assemblages. Although the experiments were designed to explore aspets of leuine inorporation as a method to measure protein synthesis by natural baterial assemblages, the work desribed here is the ontinuation of our efforts (17) to examine the regulation of transport and inorporation of amino aids by baterial assemblages. The regulation of amino aid uptake per se is important to understand beause of the role of these ompounds in the dissolved nitrogen yle and in supplying nitrogen for baterial growth. Kirhman and Hodson (17) demonstrated the tight oupling between transport of amino aids suh as leuine and their inorporation into protein. n this report we examine the biosynthesis of leuine and the utilization of leuine to synthesize other amino aids. MATRALS AND MTHODS Sampling sites. We onduted experiments at several loations to minimize the risk that our results would be appli- Downloaded from on Otober 17, 218 by guest

2 6 KRCHMAN, K'NS, AND HODSON able to only one type of aquati environment. Our initial experiments were arried out during May 1983 on the R. V. Cape Hatteras in ontinental shelf and Gulf Stream waters off the southeast oast of the United States. The preise geographial loations are given with the results of eah experiment. Water was olleted with Nisken bottles from 5 m below the surfae, unless otherwise indiated. Subsequent experiments were ompleted during Otober 1983 on the R. V. Cape Florida, whih was anhored off the oast of Andros sland, Bahamas. Subsurfae samples were olleted in polyarbonate bottles from a mangrove estuary (Fresh Creek) on Andros sland and brought bak to the Cape Florida for experimentation. n addition, Nisken bottles were used to ollet samples from S m in waters a. 1 m deep at a distane of 2 km offshore. We refer to these samples as originating from the oligotrophi Tongue of the Oean, sine leuine turnover rates and baterial abundane were low. Several experiments were onduted at Sapelo sland, Ga. Samples were olleted in polyarbonate bottles from Doboy Sound (Marsh Landing) and brought bak to the laboratory at Sapelo sland for experimentation. At all sites the experiments began within an hour after olletion of the samples. The uptake studies were onduted at the in situ temperature and in the dark. Proportion of bateria inorporating leuine. The proportion of the total baterial assemblage that took up leuine was estimated by a miroautoradiographi tehnique developed by Tabor and Neihof (3) and Meyer-Reil (21). Samples (1 ml) from the Bahamian estuary and the Tongue of the Oean were inubated with [3H]leuine for 4 h. The speifi ativity of the added leuine was 5,uCi/nmol (New ngland Nulear Corp.), and the final added onentration was.5 nm. After inubation, Formalin was added suh that the final onentration was 2%. To hek for abioti adsorption of [3H]leuine, Formalin was added before the addition of the radiolabel, and the sample was proessed with the others. The samples were stored at 4 C until analyzed. A more extensive study was onduted with samples from Sapelo sland. The onentrations used and inubation times are given below. The miroautoradiographi proedure followed was the MARG- method desribed by Tabor and Neihof (3). Briefly, appropriate sample volumes were filtered through Nulepore filters (pore size,.2,um) and rinsed one with 5 ml of filter-sterilized seawater. With the help of a safety light, the filters were transferred to aid-leaned mirosope slides that had been oated with NTB-2 autoradiographi emulsion (astman Kodak Co.). The side of the filter with the sample was plaed fae down into the emulsion. The emulsion-oated slide with the filter was then plaed onto a old surfae to solidify in total darkness. After solidifying, the slides were inubated in the dark over dessiant for 3 days at 15 C. The emulsion-oated slides with filters were developed and then stained with aridine orange as desribed by Tabor and Neihof (3). After being destained with a series of itrate buffers (3), the filters were removed by first soaking the slides in 1% glyerol for 1 min and then gently peeling the filters away from the emulsion one they were dried. The emulsion-oated slides were then examined under epifluoresene mirosopy to ount the aridine orange-stained bateria and under bright-field mirosopy to hek for aumulation of silver grains assoiated with the bateria. To be sored as a baterium that took up leuine, at least three silver grains had to be within a distane from the stained ell that was less than the length of the baterium. APPL. NVRON. MCROBOL. Total baterial abundane in eah sample was enumerated by the standard aridine orange diret-ount method (12, 34). This estimate of the total baterial abundane was ompared with the number of bateria ounted in the miroautoradiographi preparations to determine whether the bateria were ompletely transferred from the Nulepore filter to the NTB-2 emulsion. All the bateria were transferred, as total baterial abundane estimated by the regular diret-ount proedure and from the miroautoradiographi preparation differed by less than 1%. Subsamples of the Formalin-fixed seawater used for the miroautoradiographi measurements were also filtered onto membrane filters (pore size, 2,um) and rinsed. The filters, after base hydrolysis (see below), were radioassayed diretly to determine the turnover rate of leuine (assimilated radioativity divided by added radioativity) in eah sample. Possible uptake of [3H]leuine by yanobateria was also examined. The same miroautoradiographi proedure was used as desribed above, exept that the filters were not stained with aridine orange. The onentration of the added leuine was.5 nm, and the inubation time was 4 h. nstead of staining the bateria, we looked with epifluoresent and bright-field mirosopy for aumulation of silver grains assoiated with autofluoresing partiles. This experiment was onduted during May 1984 with water samples from 1 m, whih was the depth of the hlorophyll maximum, in the Gulf Stream (3 1.1' N; ' W). Reovery of [3HJleuine from protein hydrolysates. The basi proedure we propose to estimate protein synthesis is to measure the inorporation of [3H]leuine into hot TCAinsoluble material that is olleted on membrane filters. The details of our proedure is as follows. [4,5-3H]leuine and, in some experiments, unlabeled leuine or pyruvate were added to water samples. Aid-killed ontrols were also analyzed to orret for abioti uptake. The speifi ativity of the added leuine ranged from 5 to 56.5,uCi/nmol, depending on the bathes obtained from New ngland Nulear or Amersham Corp. The final onentration of the added [3H]leuine was.5 nm. At appropriate times (the preise times are given with eah experiment) subsamples were withdrawn, and TCA was added suh that the final onentration was 5%. The subsamples were extrated in a boiling water bath for 3 min. After the subsamples ooled, the TCA-insoluble material was olleted on either Nulepore or Gelman filters (pore size,.2,um) and rinsed twie with a. 3 ml of 5% old TCA. The filters were either treated for liquid sintillation ounting to measure total inorporation into the hot TCAinsoluble ell fration or frozen at -2 C for later analysis. One series of experiments examined total uptake of leuine. n that experiment the subsamples were not extrated in TCA but were filtered through membrane filters and rinsed twie with 3. ml of filtered seawater. The filters used to measure total uptake or inorporation into protein were first treated with 1. ml of Protosol (New ngland Nulear) for at least 1 h at 5 C. This treatment was effetive in minimizing self-absorption of the low-energy beta partiles, sine the same results were obtained by ombusting the filters in a Biologial Oxidizer (R. J. Harvey Co.), whih results in a homogeneous sample in the oktail. After the treatment with the alkaline regeant, the samples were neutralized with.5 ml of.5 N HCl, and 1 ml of Sintiverse (Fisher Sientifi Co.) was added. Quenh orretion was by the hannels ratio method alulated by a Bekman LS-9 sintillation ounter. We determined to what extent [3H]leuine was transformed to other ompounds, partiularly other amino aids, Downloaded from on Otober 17, 218 by guest

3 VOL. 49, 1985 LUCN NCORPORATON AS AN NDX OF PROTN SYNTHSS 61 that ould be subsequently inorporated into the hot TCAinsoluble ell fration. The following proedure was used with the Nulepore filters. The additional steps neessary for Gelman filters are desribed below. The frozen Nuleopore filters were thawed and added to 5 ml ampules along with 2 ml of 6 N HCl. After being flushed with argon or N2, the ampules were sealed and plaed in a 11 C oven for 2 h. The HCl, ontaining amino aids and other monomers resulting from the aid hydrolysis, was transferred to a glass sintillation vial (2 ml) and evaporated on a hot plate (5 C) under a stream of N2 The dried aid hydrolysate was dissolved in 5 ml of distilled water and desalted. A ation-exhange olumn was prepared with Dowex 5W-X4 (2-4 mesh; hydrogen form), whih had been rinsed several times with distilled water. After the aid hydrolysate was added to the olumn, the anioni and neutral ompounds were eluted with 5 ml of deionized water. The aqueous elution was saved and radioassayed. The amino aids were then eluted with 4 ml of 1 M NH4H. The ammonium solution, whih ontained the amino aids resulting from the aid hydrolysis, was dried down on a hot plate (5 C) under a stream of N2. The evaporated samples then were dissolved in 5 xl of 2 mm leuine. Leuine was separated from the other amino aids and monomers by a method of thin-layer hromatography (TLC) designed to separate leuine from isoleuine, valine, and phenylalanine (2). The amino aids were applied in 1-,u aliquots to silia gel TLC plates. The same volume of 2 mm leuine was spotted in an adjaent lane for every plate. We found the Whatman Linear-K plates onvenient to use beause of their preabsorbent areas and hanneled lanes. The plates were developed in a methyl ethyl ketone-pyridine-water-glaial aeti aid (73:5:2:2) solution. After hromatography, the lane ontaining the standard was sprayed with ninhydrin (.1% [wt/vol] in aetone) to visualize the leuine. The sample lanes were overed to prevent ontat with the ninhydrin. Based on the Rf of the leuine standard developed on the same plate, the leuine spots in the adjaent sample lanes were sraped and plaed in 1. ml of water in a sintillation vial. The remaining setions of the lane, inluding the preabsorbent area, were added to a different sintillation vial ontaining 1. ml of water. After the ompounds were allowed to elute off the TLC srapings for 3 min, 1 ml of Sintiverse was added. Reovery of amino aids from the sample lanes was 1%. For some experiments we sraped different areas of the sample lanes orresponding to the amino aids less polar (above the leuine spot on the hromatograph) and more polar (below the leuine spot) than leuine. Seleted amino aids, inluding isoleuine, valine, alanine, and phenylalanine, were hromatographed as standards for these experiments. We used Gelman filters in some of our experiments. When these ellulose aetate filters were hydrolyzed diretly in 6 N HCl, the resulting aramel material ould not be hromatographed. nstead, the filters were treated with the proteolyti enzyme, pepsin. First, the filters were ut in half. One half was radioassayed without pepsin treatment. The other half was plaed in 1. ml of the pepsin (1. mg/ml of.1 N HCl) for 6 h at 37 C. We found that more radioativity was solubilized from the filter with inreasing pepsin onentrations, but that onentrations higher than 1. mg ml-1 resulted in a aramel material that ould not be hromatographed. The removal of radioativity from the filter did not inrease when the filters were inubated for longer than 6 h. Pepsin had no effet on free leuine, sine we were able to reover >95% of [3H]leuine by TLC when the free amino aid was pepsin treated. After treatment the filter was radioassayed and ompared with the untreated filter to determine how muh radioativity was solubilized by the pepsin treatment. The pepsin solution, ontaining the partially digested TCA-insoluble material, was added to an ampule along with 1.6 ml of onentrated HCl to give a 6 N HCl solution. After flushing, the ampule was sealed and inubated in a 11 C oven for 2 h. The aid hydrolysate was then desalted and hromatographed as desribed above. Gelman filters and the pepsin treatment were used for experiments whose results are shown below (see Fig. 3, 4, and 7). We repeated these experiments when possible and obtained the same results. We ompared Gelman and Nuleopore filters diretly and found that the measured proportion of total 3H-radioativity reovered as [3H]leuine was the same for both filters. Furthermore, both filters are equally effetive in trapping radioativity in the hot TCA-insoluble fration when used for samples from natural aquati environments. Nulepore filters were not as effetive as Gelman filters in trapping hot TCA preipitates from high ell densities of. oli. norporation of [14C]pyruvate into protein. To investigate the biosynthesis of leuine, we examined the inorporation of [3-_4C]pyruvate (New ngland Nulear) into the hot TCA-insoluble fration. The speifi ativity of the added pyruvate was 15 mci/mmol. The experimental protool was similar to that desribed above for [3H]leuine inorporation. After inubation of the water sample with [14C]pyruvate, the samples were extrated in hot 5% TCA for 3 min. The hot TCA-insoluble material was olleted on filters, whih were either radioassayed diretly or frozen for later TLC analysis. The transformation of [14C]pyruvate to [14C]leuine was examined by hydrolyzing the filters in 6 N HCl and separating the amino aids by TLC as desribed for [3H]leuine inorporation. The proportion of [14C]pyruvate used for leuine biosynthesis was estimated from the amount of [14C]leuine reovered ompared with the radioativity sampled from the remaining setions of the TLC plate. We tested whether the presene of extraellular leuine inhibited the biosynthesis of leuine. The proportion of [14C]pyruvate reovered as [14C]leuine from the hydrolyzed TCA-insoluble material was measured after inubation with different onentrations of nonradioative leuine along with the [14C]pyruvate. The nonradioative leuine was added just before addition of the [14C]pyruvate. The inubation times and onentration of [14C]pyruvate are given for eah experiment below. Aid-killed ontrols were also analyzed and used to orret for abioti uptake. One experiment tested for the effet of leuine on total uptake of [14C]pyruvate. n that experiment the subsamples were filtered, rinsed twie with 3 ml of filter-sterilized seawater, and radioassayed. Data analysis. Several experiments measured the inorporation into protein of [3H]leuine in the presene of different onentrations of nonradioative leuine. Sine this is analogous to the approah of Wright and Hobbie (36), we alulated the maximum inorporation rate (Vmax) by using standard nonlinear parameter estimation tehniques (18), whih fit the equation of Wright and Hobbie diretly to the data without linear transformation. The lines drawn were fit by eye. RSULTS Several parameters must be examined to demonstrate that a hot TCA extration after inubation of a water sample with Downloaded from on Otober 17, 218 by guest

4 62 KRCHMAN, K'NS, AND HODSON Proportion of baterial assemblages that inorporated leuine in three marine environments' No. of % nvironment [3H]leuine Turnover bateria dpm Leuineonn (nm) (% h-1) ml-' ell-' ative (x 15) bateria TABL 1. Tongue of the Oean Bahamian estuary Sapelo sland "The inubation time was 2 h for samples from the Tongue of the Oean and the Bahamian estuary. The inubation time for samples from Sapelo sland was 4 h. The interpolated values for the perentage of leuine-ative bateria in 2-h samples are 23% for.5 nm leuine and 53% for 1 nm leuine. This interpolation is based on data shown in Fig. 1. [3H]leuine is an adequate method for measuring the rate of protein synthesis by bateria in natural mirobial assemblages. Here we examined the following parameters: (i) the proportion of the total baterial assemblage that assimilates leuine, (ii) the degree of leuine degradation to other ompounds inorporated into the hot TCA-insoluble fration, and (iii) de novo synthesis of leuine. Proportion of bateria inorporating leuine. We used miroautoradiography (3) to measure the proportion of baterial ells that took up leuine (leuine-ative bateria) in a Bahamian estuary, in the salt marsh estuary at Sapelo sland, and in the oligotrophi Tongue of the Oean. The proportion was high (>5%) for all three environments (Table 1). n fat, when.5 nm of [3H]leuine was added, a greater proportion of ells was labeled with silver grains in the Tongue of the Oean than in the estuaries, even though baterial abundane and turnover rates of leuine were greater and faster in the estuaries. No bateria with three assoiated silver grains were observed in the Formalin-killed ontrols. The measured proportion of leuine-ative ells depends on several fators (7, 3). We found with samples from Sapelo sland that the proportion of leuine-ative ells was 3% when.5 nm [3H]leuine was added but 63% with the addition of 1 nm (Table 1). The proportion of leuine-ative bateria also inreased with inubation time (Fig. 1). The proportion was 23% at 3 min and 63% at 4 h when 1 nm [3H]leuine was added (Fig. 1). n addition, samples with the greatest amount of assimilated radioativity per ell had the highest measured proportion of leuine-ative ells (Table 1). Although the biologial signifiane of the variation in the proportion of leuine-ative bateria with different proedures is unertain, it is lear that most bateria take up leuine in environments that differ greatly in total baterial abundane and leuine turnover rates. We observed no evidene of leuine uptake by phytoplankton or yanobateria. Only 1 of more than 1 phytoplankton examined had any assoiated silver grains. This result is onsistent with several studies using miroautoradiography (7, 13), size frationation (1), and antibiotis (17), whih all indiate the predominane of proaryotes as sinks for the nanomolar onentrations of leuine and other amino aids typially measured in natural waters. Likewise, we observed no silver grains assoiated with the ooid yanobateria, although our observations are based on samples from only one Gulf Stream loation. Cyanobateria and phytolankton an take up amino aids in pure ulture (2, 11), but our results indiate that heterotrophi bateria outompete autotrophi organisms for the nanomolar onentrations of leuine added in these experiments. Leuine inorporation into protein and leuine degradation. norporation of leuine into a hot TCA-insoluble ell fration would not be a good measure of protein synthesis if leuine were primarily degraded to other ompounds that were subsequently inorporated into the hot TCA-insoluble fration. f degradation of leuine were important, then the rate of protein synthesis would be overestimated to the degree that radiolabeled amino aids and other by-produts resulting from leuine degradation are inorporated into the hot TCA-insoluble fration. Bateria in pure ulture typially inorporate leuine into protein without prior transformation of leuine to other amino aids (29). Usually we found this to be true also for natural baterial assemblages. We observed that a high proportion (a. 9%) of the leuine taken up was inorporated into protein and that this proportion did not vary with time in experiments with baterial assemblages from the salt marsh at Sapelo sland (Fig. 2). Nearly all of the radioativity inorporated into protein ould be reovered as [3H]leuine when the protein fration was hydrolyzed and the amino aids were separated by TLC. The proportion of total radioativity reovered as leuine did not vary signifiantly during a 7-h inubation (Fig. 2). These results demonstrate that relatively little ( to 2%) of the assimilated leuine was transformed to other amino aids before inorporation into protein. - L ) C ) 1-J- [Leu] =.5nM lonm %/OLeu Ative * Total Uptake nubation Time (h) APPL. NVRON. MCROBOL o rc 4 > (D 3 C O. 2 a. FG. 1. Measured perentage of the baterial assemblage that assimilated [3H]leuine (% leu-ative bateria) as determined by miroautoradiography. The onentrations of the added [3H]leuine are given in the key. The experiment was onduted at Sapelo sland in February Downloaded from on Otober 17, 218 by guest

5 VOL. 49, 1985 LUCN NCORPORATON AS AN NDX OF PROTN SYNTHSS 63 We next tested whether the onentration of leuine affeted the degradation of leuine to other amino aids. The proportion of radioativity reovered as leuine did not hange substantially with the addition of up to 5 nm of leuine to samples from ontinental shelf water (Fig. 3). The proportion of total TCA-insoluble radioativity reovered as leuine in samples from the Bahamian estuary also did not vary signifiantly with leuine additions (data not shown). Leuine degradation appears to be muh greater in lessprodutive aquati systems. The proportion of inorporated radioativity reovered as leuine was substantially lower in a Gulf Stream sample (Fig. 4) than in the less oligotrophi waters of the ontinental shelf and at Sapelo sland (Fig. 2). One measure of the differene between the Gulf Stream and shelf water samples that we examined is the maximum inorporation rate of leuine (36). The maximum rate was nmol liter-1 h-' in shelf waters with little leuine degradation (data not shown) and.166 ±.4 nmol liter-' h-1 in Gulf Stream samples with 49% leuine degradation (Fig. 4). The addition of leuine had no lear effet on the proportion reovered, although it was 97% with the addition of 5 nm to the Gulf Stream sample (Fig. 4). Similar results were obtained when the experiment was repeated with samples from the oligotrophi Tongue of the Oean; the proportion reovered as leuine was 39% (Fig. 5). Our preliminary results suggest that the radioativity not assoiated with leuine was assoiated with ompounds more polar than leuine. These polar ompounds inlude valine, a likely byprodut of leuine degradation (23). This omparison among environments suggests that 14 C y CD -J Q- 1 v J -9 M nubation Time (Minutes) FG. 2. Total uptake and inorporation into protein of [3H]leuine (Leu) and the proportion of inorporated radioativity reovered as [3H]leuine. The data given for total uptake and inorporation into protein are the means of two repliates. The experiment was onduted at Sapelo sland in February r _ 1.2 j - a:-._ O.8. Q.. o 3.6,1 OA -* _* 9 *--@% RCOVRD AS L -o TOTAL NCORPORATON Li s) ) a, i- [Leuine] nm 1 8 CD 7 r the FG. 3. Leuine (Leu) inorporation rate and proportion of inorporated radioativity reovered as [3H]leuine. The given onentration of leuine is that of the added nonradioative leuine, -e whih does not inlude.5 nm [3H]leuine. The inubation time was ; 1. h. The loation of this experiment was the ontinental shelf of m the southeast United States (29 1.3' N; 814.5' W). rcd a.f supply of organi arbon may determine how muh leuine is inorporated diretly into protein and how muh is degraded to other amino aids. To test this hypothesis diretly, we preinubated Tongue of the Oean samples for 3 min in 1,uM pyruvate. This ompound was seleted beause it is a preursor for leuine biosynthesis (33). We hypothesized that if leuine degradation were dependent on the rate of arbon supply, then the addition of pyruvate should derease leuine degradation and inrease the reovery of [3H]leuine from protein. A signifiantly higher proportion of the exogenous [3H]leuine was in fat inorporated intat into protein in the presene of pyruvate than in the absene of pyruvate (P <.5; the signed-rank test of Wiloxon) (Fig. 5). With the addition of pyruvate, 49% more radioativity, whih originated as [3H]leuine, was reovered as leuine after aid hydrolysis of the isolated protein. Pyruvate had no signifiant effet on the maximum inorporation rate of leuine, whih was.16 ±.3 nmol liter-' h-v in this experiment. n short, the addition of pyruvate redued leuine degradation, whih supports the hypothesis that the degree of leuine degradation is dependent on the organi arbon supply. A omplete budget of the inorporated radioativity was ompiled for every experiment. The average reovery for the experiments with [3HJleuine was 67%. We were onerned that the radioativity unaounted for may somehow be assoiated with by-produts of leuine degradation and that we were underestimating leuine degradation. Several ontrol experiments were performed to rule out this Downloaded from on Otober 17, 218 by guest

6 64 KRCHMAN, K'NS, AND HODSON APPL. NVRON. MCROBOL r.? o -21 5L V --e* % RCOVRD AS LU o---o TOTAL NCORPORATON \ 1, /.A [Leuinel, nm OC FG. 4. Leuine (Leu) inorporation rate and proportion of inorporated radioativity reovered as [3H]leuine. The given onentration of leuine is that of the added nonradioative leuine, whih does not inlude.5 nm [3H]leuine. The inubation time was 1. h. The loation of this experiment was the westem boundary of the Gulf Stream ( ' N; ' W). possibility. To determine whih step(s) in our proedure resulted in the loss of radioativity, [3H]leuine was subjeted to the aid hydrolysis, desalting, and the htomatographi proedures used to examine 3H-radioativity inorporated into the TCA-insoluble fration. The reovery of [3H]leuine in these ontrol experiments was similar (7%) to the reovery obtained during typial experiments. The reovery for eah step in our proedures was tested and found to be aeptable (>9% reovery). t appears that most of the loss in radioativity omes from transferring the samples from one step of the proedure to the next. The small loss (<1%) at eah step aumulates. There was little loss of radioativity (<5%) from the evaporation of volatile ompounds or from the aqueous elution of the desalting olumn, indiating that the prodution of 3H2 (from the degradation of non-leuine amino aids during our proedures) and 3H-sugars (whih would result from the hydrolysis of any polysaharides that may have preipitated in hot TCA) was insignifiant. nhibition of leuine biosynthesis by extraellular leuine. Rates of protein synthesis would be underestimated based on the rate of leuine inorporation if de novo synthesis of leuine were signifiant. n pure ultures of bateria, the addition of leuine inhibits the biosynthesis of leuine (33). To test whether extraellular leuine bloks de novo synthesis of leuine by baterial assemblages in marine environments, we examined the effet of leuine on the inorporation into protein of [14C]pyruvate, whih is a preursor for leuine biosynthesis (33). n samples from the Bahamian = 8 -J n = a a 6 -a 4 _ o CD C r. o-o WTH PYRUVAT _- -WTHOUT PYRUVAT [Leuinel, nm FG. 5. Proportion of [3H]leuine inorporated into protein that was p reovebred as [3H]leuine with and without the addition of pyruvate. The lines show the averages with and without the addition of pyruvate, The proportion reovered as [3H]leuine was signifi- with than without the addition of pyruvate (P <.5; antly highezr the signed-r; *ank test of Wiloxon). The inubation time was 3 h. This experiment was onduted with Tongue of the Oean water. estuary, inorporation of ['4C]pyruvate into protein was not affeted by additions of leuine up to 3 nm (Fig. 6). When 5 nm was added, inorporation dereased by 25%; the addition of 2 nm leuine inhibited pyruvate inorporation by 4% (Fig. 6). Similarly, in samples from the Tongue of the Oean, inorporation dereased by 53% when 3 nm of leuine was added (Fig. 6). These results suggest that - 8r m6 L -CZ W * ''2 Leuinel, nm FG. 6. norporation rate of [14C]pyruvate into protein in the presene of added leuine. The experiment was onduted simultaneously with Tongue of the Oean water (losed irles) and water from a Bahamian estuary (open irles). The data on inorporation rates from the Tongue of the Oean should be divided by a fator of 1. The added onentration of pyruvate was 2 FM, and the inubation time was 2 h. /._l Downloaded from on Otober 17, 218 by guest

7 VOL. 49, 1985 LUCN NCORPORATON AS AN NDX OF PROTN SYNTHSS 65 extraellular leuine does inhibit leuine biosynthesis by these marine baterial assemblages. The addition of leuine apparently inhibited leuine biosynthesis to a greater extent than the synthesis of other amino aids deriving arbon from pyruvate. f synthesis of all the pyruvate family amino aids were inhibited equally by leuine, then the proportion of radioativity, whih originated as [14C]pyruvate, reovered as [14C]leuine should not be affeted by the addition of leuine. n ontrast, in samples from the Sapelo sland estuary in August 1983, the proportion reovered as ['4C]leuine was 3% with no leuine addition and dereased to 5% with the addition of 3 nm leuine (Fig. 7). Total inorporation of ["4C]pyruvate into the protein fration dereased by nearly 4% with the addition of 3 nm leuine (Fig. 7). These results indiate that the addition of leuine inhibited leuine biosynthesis more so than the de novo synthesis of other amino aids. The effet of leuine on [14C]pyruvate uptake and inorporation was examined again at Sapelo sland in February As observed with the August samples, the proportion reovered as [14C]leuine after aid hydrolysis dereased from 23 to 12% with the addition of 1 nm leuine (Table 2). Total uptake of pyruvate dereased by only 4% with the addition of 1 nm leuine, whereas the proportion of total uptake that was inorporated into protein dereased from 95 to 84% (Table 2). We onlude that the addition of leuine does not affet pyruvate transport, but does inhibit de novo synthesis of amino aids (primarily leuine) from pyruvate by marine baterial assemblages. - -._ CL- a) 1- ~ 1.4r 1.3k 1.2F _--4 % RCOVRD AS LU -O TOTAL NCORPORATON k.91.8k N%. -. -% ~~~ [Leuine], nm od2o FG. 7. norporation rate of ['4C]pyruvate into protein and the proportion of ['4C]pyruvate inorporated into protein that was reovered as [14C]leuine (Leu) in the presene of added leuine. The experiment was onduted at Sapelo sland in August The added onentration of pyruvate was 8 nm, and the inubation period was 1 h. TABL 2. ffet of leuine on ["4C]pyruvate uptake, inorporation, and biosynthesis of leuinea Added leuine Total uptake % % (nm) (kdpm ml-') norporation Reovered into protein' as leuine' a Samples were taken from Sapelo sland in February The inubation time was 1 h at the in situ temperature of 18 C. The added onentration of pyruvate was 4,uM. b The perentage given is the proportion of total uptake (no frationation) that was insoluble in hot TCA. ' The perentage reovered as leuine was determined by hydrolyzing hot TCA-insoluble extrats in 6 N HC1 and isolating [14Cjleuine by TLC. The perentage reovered as leuine is the amount of radioativity reovered as leuine divided by the total amount of radioativity on the TLC plate. All values are the means of repliates. DSCUSSON Our results indiate that the inorporation of [3H]leuine into the hot TCA-insoluble ell fration an be used as an index of protein synthesis by baterial assemblages in natural waters. Most bateria assimilated leuine in the marine environments we examined. Other studies had demonstrated that a high proportion of bateria (>5%) in natural assemblages assimilate at least some omponents of a mixture of amino aids (7, 13, 3). Biosynthesis of leuine is inhibited by the addition of nanomolar onentrations of leuine, whih does not appear to hange the rate of protein synthesis. The rate of baterial protein synthesis and growth is most likely limited by more than just the supply of leuine. We observed linear inorporation rates of leuine during our inubation periods, suggesting that protein synthesis was not stimulated during our experiments (Fig. 1). f protein synthesis ould be stimulated during a short-term inubation by the addition of leuine, then other amino aids would have to be synthesized from leuine. ven with relatively high onentrations of leuine (1 nm), baterial assemblages do not use leuine to synthesize other amino aids (Fig. 2 and 3), exept in oligotrophi environments (Fig. 4). t is not possible presently to assess quantitatively the rate of leuine biosynthesis remaining after the addition of extraellular leuine. However, our data indiate that muh of leuine biosynthesis is inhibited by the addition of leuine. t is true that [14C]leuine was reovered from the inorporation of [14C]pyruvate into protein, even though the onentration of extraellular leuine was relatively high (Fig. 7; Table 2). However, leuine biosynthesis may be overestimated in these experiments beause of the large addition (as muh as 4 p.m) of [14C]pyruvate. Beause the speifi ativity of available [14Clpyruvate is low, high onentrations have to be used to obtain measurable rates. n spite of the high pyruvate addition, nanomolar onentrations of extraellular leuine redued total [14C]pyruvate inorporation into protein by 3 to 53% (Fig. 6 and 7). The maximum expeted derease in this inorporation rate is a. 29%, based on the theoretial and atual perentage of inorporated [14C]pyruvate reovered as [14C]leuine when no leuine is added (a relative measure of leuine biosynthesis). Thus, muh of leuine biosynthesis does appear to be inhibited by the addition of leuine. Determining the rate of leuine biosynthesis is one part of the larger problem in estimating the speifi ativity of intraellular pools of leuine and leuyl-trna. t is nees- Downloaded from on Otober 17, 218 by guest

8 66 KRCHMAN, K'NS, AND HODSON sary to know the speifi ativity of the leuyl-trna pool to diretly alulate the rate at whih [3H]leuine is inorporated into protein (19). For a number of reasons this speifi ativity would be diffiult, if not impossible, to estimate for baterial assemblages ourring in most aquati systems. t is doubtful that the pools in those miroorganisms inorporating leuine (primarily bateria) ould be distinguished from the pools in miroorganisms not inorporating leuine. Phytoplankton, whih do not take up signifiant amounts of amino aids (1, 7, 17) but whih have amino aids pools (35), undoubtedly have larger internal leuine and leuyl-trna pools than the bateria, as the phytoplankton biomass is muh larger than the baterial biomass in the euphoti zone of aquati systems. To irumvent the speifi ativity problem, we have been using an approah based on the suggestions of Moriarty and Pollard (24) and Kirhman et al. (16) to measure a onversion fator, whih relates the inorporation rate of leuine to the rate of protein synthesis. The results will be reported in more detail elsewhere (D. Kirhman and R. Hodson, manusript in preparation). Briefly, grazing ativity by baterivores, whih usually prevents baterial abundane from inreasing signifiantly, is redued by adding euaryoti inhibitors (25) or by diluting the mirobial assemblage with filtered water (6, 16). Baterial abundane and biomass inrease over time after the grazing ativity has been redued, if not eliminated. At various times subsamples are withdrawn, and the inorporation rate of leuine is measured as desribed by Moriarty and Pollard (24); the inorporation rate of [3H]leuine is measured in the presene of different onentrations of nonradioative leuine. A onversion fator is then alulated by using the equations desribed by Kirhman et al. (16). n addition to variation in the speifi ativity of leuyltrna, another potential problem in using the rate of leuine inorporation to alulate rates of protein synthesis is possible variation in the amount of leuine in baterial protein. However, the molar fration of leuine in a wide variety of proteins is relatively onstant. Reek and Fisher (28) ompiled the reported values for the amino aid ontent of 27 proteins. The leuine molar fration of prokaryoti enzymes is 8.8% (standard deviation [SD] = 2.1%); the leuine ontent of euaryoti enzymes is 8.2% (SD = 2.%). The average leuine molar fration given in a more reent ompilation of 61 baterial proteins is 8.8% (SD = 2.%) (27). These averages agree well with the best estimate of the leuine ontent in natural baterial assemblages. The data of Hagstrom et al. (8) indiate that the leuine molar fration of a marine baterial assemblage in ontinuous ulture with unamended seawater was 9.1 and 8.3% at dilution rates of.18 h-1 and.11 h-1, respetively. The important point is that, based on the extensive data set on leuine ontent of proteins studied in the laboratory, 95% of all proteins will have a leuine ontent that varies on the average less than.3% (a onfidene interval based on n = 27 and SD = 2.1%) from the mean of 8.8%. Thus, variation in leuine ontent is very unlikely to ause signifiant variation in rates of leuine inorporation and hene in the alulated rate of protein synthesis by natural baterial assemblages. Beause protein is a large proportion of baterial biomass (a. 5%), the rate of protein synthesis should be a large fration of the rate of total baterial prodution. Rates of leuine inorporation an be used to estimate baterial biomass prodution; the rate of leuine inorporation is divided by the proportion of leuine in protein (8.8%) and the amount of protein in baterial ells (a. 5%). However, APPL. NVRON. MCROBOL. estimates of baterial prodution based on protein synthesis (leuine inorporation) will equal estimates of prodution based on DNA synthesis (thymidine inorporation; see referenes 6 and 7) only during balaned growth. Rates of protein and DNA synthesis, normalized to the amount of eah maromoleule per ell, are equal during balaned growth (14, 26). Whether the baterial assemblage is in balaned or unbalaned growth is an important question to address. The proportion of primary prodution proessed by bateria has been estimated from rates of DNA synthesis and by assuming that the uptake of organi ompounds is losely oupled to the inorporation of [3H]thymidine (5, 6), whih is the ase during balaned growth. Given that the onditions for baterial growth hange on small temporal and spatial sales, it is unlear what perentage of the time baterial assemblages are in balaned growth. Although some investigators have onluded that baterial assemblages in aquati systems are in balaned growth (3, 9, 17), we believe that omparing rates of thymidine and leuine inorporation is a diret approah for examining the extent of balaned versus unbalaned growth. We have observed signifiant temporal and spatial differenes between rates of [3H]thymidine inorporation into DNA and rates of [3H]leuine inorporation into protein (D. Kirhman, R. Murray, L. Pomeroy, and R. Hodson, manusript in preparation). n addition to eluidating how well leuine inorporation measures baterial protein synthesis, the results disussed here inrease our understanding of the relationships among leuine uptake, leuine biosynthesis, and inorporation into protein. Our results demonstrate that baterial assemblages an quikly regulate the relative rates of leuine degradation and biosynthesis in response to the addition of organi ompounds. When pyruvate was added, the amount of leuine degraded to other amino aids dereased on a time sale shorter than the presumptive growth rate of the bateria. Similarly, leuine biosynthesis dereased quikly (<1 h) when leuine was added. n both ases, the bateria apparently maximize their utilization of extraellular leuine for protein synthesis, rather than synthesizing leuine de novo or utilizing leuine for the synthesis of other amino aids. xperiments suh as those desribed here give insight into the regulation of baterial metabolism in response to hanges in the dissolved organi arbon pool. ACKNOWLDGMNTS We thank D. Montiello for his advie on methods to examine leuine metabolism. We also thank L. Pomeroy for allowing us to partiipate on ruises of the Cape Hatteras. This work was supported in part by National Siene Foundation grants BSR and OC and by National Oeanographi and Atmospheri Administration sea grant NA 8AA-D- 91. LTRATUR CTD 1. Azam, F., and R.. Hodson Size distribution and ativity of marine miroheterotrophs. Limnol. Oeanogr. 22: Chapman, J. S., and J. C. Meeks Glutamine and glutamate transport by Anabaena variabilis. J. Bateriol. 156: Cuhel, R. L., H. W. Jannash, and C. D. Taylor Mirobial growth and maromoleular synthesis in the northwestern Atlanti oean. Limnol. Oeanogr. 28: Cuhel, R. L., C. D. Taylor, and H. W. Jannash Assimilatory sulfur metabolism in marine miroorganisms: onsiderations for the appliation of sulfate inorporation into protein as a measurement of natural population protein synthesis. Appl. nviron. Mirobiol. 43: Downloaded from on Otober 17, 218 by guest

9 VOL. 49, 1985 LUCN NCORPORATON AS AN NDX OF PROTN SYNTHSS Duklow, H. W., and D. Kirhman Baterial dynamis and distribution during a spring diatom bloom in the Hudson River Plume. J. Plankton Res. 5: Fuhrman, J. A., and F. Azam Baterioplankton seondary prodution estimates for oastal waters of British Columbia, Antartia, and California. Appl. nviron. Mirobiol. 39: Fuhrman, J. A., and F. Azam Thymidine inorporation as a measure of heterotrophi baterioplankton prodution in marine surfae waters: evaluation and field results. Mar. Biol. 66: Hagstrom, A., J. A. Ammerman, S. Henrihs, and F. Azam. Baterioplankton growth in seawater.. Organi matter utilization during steady-state growth. Mar. ol. Prog. Ser. 18: Hanson, R. B., and H. K. Lowery Nulei aid synthesis in oeani miroplankton from the Drake Passage, Antartia: evaluation of steady-state growth. Mar. Biol. 73: Hanson, R. S., and J. A. Phillips Chemial omposition, p n P. Gerhardt, R. G.. Murray, R. N. Costilow,. W. Nester, W. A. Wood, N. R. Kreig, and G. B. Phillips (ed.), Manual of methods for general bateriology. Amerian Soiety for Mirobiology, Washington, D.C. 11. Hellebust, J. A., and J. Lewin Heterotrophi nutrition, p n D. Werner (ed.), The biology of diatoms. University of California Press, Berkeley. 12. Hobbie, J.., R. J. Daley, and S. Jasper Use of Nulepore filters for ounting bateria by fluoresene mirosopy. Appl. nviron. Mirobiol. 33: Hoppe, H. G Determination and properties of atively metabolizing heterotrophi bateria in the sea, investigated by means of miroautoradiography. Mar. Biol. 36: ngraham, J. L.,. Maal$e, and F. C. Neidhardt Growth of the baterial ell. Sinauer Assoiation, Sunderland, Mass. 15. Jordan, M. J., and G.. Likens Measurement of planktoni baterial prodution in an oligotrophi lake. Limnol. Oeanogr. 25: Kirhman, D., H. W. Duklow, and R. Mithell stimates of baterial growth from hanges in uptake rates and biomass. Appl. nviron. Mirobiol. 44: Kirhman, D., and R. Hodson nhibition by peptides of amino aid uptake by baterial populations in natural waters: impliations for the regulation of amino aid transport and inorporation. Appl. nviron. Mirobiol. 47: Li, W. K. W Consideration of errors in estimating kineti parameters based on Mihaelis-Menten formalism in mirobial eology. Limnol. Oeanogr. 28: Matin, A. F., and M. Rabinowitz Measurement of half-life of rat ardia myosin heavy hain with leuyl-trna used as preursor pool. J. Biol. Chem. 252: MBride, R. W., D. W. Jolly, B. M. Kadis, and T.. Nelson Rapid thin-layer hromatographi separation of isoleuine, leuine, and phenylalanine. J. Chromatogr. 168: Meyer-Reil, L. A Autoradiography and epifluoresene mirosopy ombined for the determination of number and spetrum of atively metabolizing bateria in natural waters. Appl. nviron. Mirobiol. 36: Mitra, R. S Protein synthesis in sherihia oli during reovery from exposure to low levels of Cd2+. Appl. nviron. Mirobiol. 47: Montiello, D. J., and R. N. Costilow. nteronversion of valine and leuine by Clostridium sporogenes. J. Bateriol. 152: Moriarty, D. J. W., and P. C. Pollard DNA synthesis as a measure of baterial produtivity in seagrass sediments. Mar. ol. Prog. Ser. 5: Newell, S. Y., B. F. Sherr,. B. Sherr, and R. D. Fallon Baterial response to presene of eukaryote inhibitors in water from a oastal marine environment. Mar. nviron. Res. 1: Prithard, R. H., and D. W. Tempest Growth: ells and populations, p n J. Mandelstam, K. Mquillen, and. Dawes (ed.), Biohemistry of baterial growth. John Wiley & Sons, n., New York. 27. Reek, G. R Amino aid ompositions of seleted proteins, p n A.. Laskin and H. A. Lehevalier (ed.), Handbook of mirobiology, vol. 2. Mirobial omposition. CRC Press, n., Boa Raton, Fla. 28. Reek, G. R., and L. Fisher A statistial analysis of the amino aid ompositions of proteins. nt. J. Peptide Protein Res. 5: Roberts, R. B., D. B. Cowie, R. H. Abelson,. T. Bolton, and R. J. Britten Studies of biosynthesis in sherihia oli. Carnegie nstitution of Washington publiation no. 67. Carnegie nstitute of Washington, Washington, D.C. 3. Tabor, P. S., and R. A. Neihof mproved miroautoradiographi method to determine individual miroorganisms ative in substrate uptake in natural waters. Appl. nviron. Mirobiol. 44: Tenore, K. R., R. B. Hanson, B.. Dornseif, and C. N. Wiederhold The effet of organi nitrogen supplement on the utilization of different soures of detritus. Limnol. Oeanogr. 24: Turnbough, C. L Regulation of sherihia oli aspartate transarboamylase synthesis by guanosine tetraphosphate and pyrimidine ribonuleoside triphosphates. J. Bateriol. 153: Umbarger, H Amino aid biosynthesis and its regulation. Annu. Rev. Biohem. 47: Watson, S. W., T. J. Novitsky, H. L. Quinby, and F. W. Valois Determination of baterial number and biomass in the marine environment. Appl. nviron. Mirobiol. 33: Wheeler, P. W Phytoplankton nitrogen metabolism, p n. Carpenter and D. Capone (ed.), Nitrogen in the marine environment. Aademi Press, n., New York. 36. Wright, R. T., and J.. Hobbie Uptake of organi solutes in lake water. Limnol. Oeanogr. 1: Yamamori, T., K. to, Y. Nakamura, and T. Yura Transient regulation of protein synthesis in sherihia oli upon shift-up of growth temperature. J. Bateriol. 134: Downloaded from on Otober 17, 218 by guest