MARINE ECOLOGY PROGRESS SERIES Published November 3 Mar Ecol Prog Ser
Nitrogen fixation, uptake and metabolism in natural and cultured populations of Trichodesmiumspp.
Margaret R. Mulholland* , Douglas G. Capone*"
Chesapeake Biological Laboratory, University of Maryland Center for Environmental Science, PO Box 38. Solomons, Maryland 20688, USA
ABSTRACT: Uptake rates of several combined N sources, NZ fixation, intracellular glutamate (glu) and glutamine (gln) pools, and glutamine synthetase (GS) activity were measured in natural populations and a culture of Trichodesmium IMSlOl grown on seawater medium without added N. In cultured populations, the ratio of GS transferase/biosynthetic activity (an index of the proportion of the GS pool that is active) was lower, and intracellular pools of glu and gln and the ratios of gldglu and glnla- ketoglutarate (glnlakg) ratios were higher when NZ fixation was highest (mid-day). There was an excess capacity for NH,' assimilation via GS, indicating that this was not the rate-limiting step in N uti- lization. In natural populations of Trichodesmium spp., the gldglu ratio closely approximated the glnlakg ratio over the die1 cycle. High gln/glu and gln/akg ratios were noted in near-surface popula- tion~. These ratios decreased in samples collected from greater depths. Natural populations of Tn- chodesmium spp. showed a high capacity for the uptake of NH4+,glu, and mixed amino acids (AA). Rates of NO3- and urea uptake were low. NH,+ accumulated in the culture medium during growth and rates of NH4+ uptake showed a positive relationship with the NH4+ concentration in the medium. Although rates of NZ fixation were highest and accounted for the majority of the total measured N uti- lization during mid-day, rates of NH4+uptake exceeded rates of NZfixation throughout much of the die1 cycle. In exponentially growing cultures, only 23% of the total daily N utilization was due to N, fixa- tion while NH,+ uptake accounted for more than 70%. Based on N2 fixation alone, the N turnover time for this culture during exponential growth was on the order of 9 d. This is consistent with the observed chlorophyll-based growth rates for these cultures suggesting that N, fixation was responsible for net growth. Our results contrast with the view that natural populations of Trichodesmium spp. acquire their cell N exclusively through N, fixation. C productivity may overestimate N demand for net pro- duction if regenerated production is significant in these populations.
KEY WORDS: Mchodesmium . Nitrogen metabolism . N2fixation . Amino acid pools . Nitrogen uptake
INTRODUCTION aggregates that are spherical (puffs) or fusiform (tufts). Colonies and free trichomes are capable of fixing NZ, which allows them to alleviate N limitation in the seas Trichodesmium spp. are common throughout the in which they occur. tropical and subtropical oligotrophic ocean (Capone et In natural populations of Trichodesmium thiebautii al. 1997). They occur as free trichomes or as colonial nitrogenase synthesis and activity exhibit a daily cycle (Capone et al. 1990, Zehr et al. 1993). Nitrogenase, the Present addresses: enzyme complex responsible for catalyzing the reduc- 'Marine Sciences Research Center, SUNY Stony Brook, tion of NZ,activity is confined to the daylight hours and Stony Brook, New York 11794-5000, USA. E-mail: [email protected] rates of NZ fixation are highest around mid-day, sug- "Department of Biological Sciences, University of Southern gesting that light or photosynthesis might regulate California, Los Angeles, California 90089, USA nitrogenase activity (Saino & Hatton 1978, Capone et
Q Inter-Research 1999 Resale of fullarticle not perrmtted Mar Ecol Prog Ser 188: 33-49, 1999
al. 1990). Chen et al. (1996) established in cultures of 1988). A capacity for glu and gln uptake by colonial Trichodesm~um IMSlOl that this cycle is driven by an aggregates was demonstrated in natural populations of endogenous rhythm. Trichodesmium spp. (Saino 1977, Carpenter et al. N metabolism In natural populations of Tricho- 1992, Capone et al. 1994). desmium spp. has been shown to proceed via the glut- We therefore examined the ability of Trichodesmjum amine synthetase (GS)/glutamate synthase (GOGAT) to assimilate combined N in cultures grown on medium pathway (Carpenter et al. 1992), as for most dia- without added N and in natural populations in order to zotrophic cyanobacter~a. GS activity was measured in eval.uate the generalization that they are ~iholly populations of Tnchodesmium spp. from the North dependent upon NZ fixation and also to understand Atlantic using the transferase assay. Rates did not vary how the observed patterns of nitrogenase synthesis, significantly over a diel cycle although concentrations activity and modification are regulated. Both the of GS protein were positively correlated with concen- gln/glu ratio and the gln/a-ketoglutarate (gln/cr.kg) trations of nitrogenase protein in immuno-labeling ratios were measured in the same samples to deter- studies (Carpenter et al. 1992). Cellular nitrogenase mine whether these ratios were comparable and and GS concentrations both declined at night when whether these metabolites could be used to predict cells were not fixing N2. patterns of N utilization and metabolism. GS activity Little is known about the growth cycle, the phys- was measured using the transferase assay, as well as iological status or the regulation of NZ fixation in the forward reaction assay, to determine the in vivo natural populations of Trichodesmium spp. The gluta- potential for NH,' assimilation relative to the diel pat- mine/glutamate (gln/glu) ratio has been used as an tern of N2 fixation. By characterizing the cellular bio- index of N-status in phytoplankton and cyanobacteria chemical and physiological conditions under which NZ (Flynn et al. 1989, Flynn 1990, Flynn & Gallon 1990). fixation and N uptake occur, we hoped to identify fac- Capone et al. (1994) found that intracellular glu and tors contributing to the control and regulation of these gln concentrations and the ratio of gln/glu varied on a processes in Trichodesmium spp. diel basis in Trichodesmium spp., in parallel with the pattern of N2 fixation. N2 fixation is generally held to be the primary mode METHODS of N acquisition for these species in nature (Carpenter & McCarthy 1975, Mague et al. 1977, Carpenter 1983b, Sample collection. Concentrations of intracellular Carpenter et al. 1997). While surface waters in the metabolite pools of glu, gln and a-ketoglutarate and tropical oligotrophic ocean gyres are generally de- rates of N, fixation, N uptake, and GS activity were pleted in combined N, NH,' and dissolved organic N measured in natural assemblages of Trjchodesmium (DON) are rapidly recycled to support much of the spp. froin the tropical North Atlantic Ocean during apparent N demand of primary production in these cruises aboard the RV 'Gyre' in May 1994 and the RV systems (Eppley & Peterson 1979, Bronk et al. 1994). 'Seward Johnson' in April 1996, and the Eastern Trichodesmium spp. contributes directly to NHdt turn- Caribbean Sea during cruises aboard the RV 'Seward over in these systems by its release of amino acids Johnson' in January 1995 dnd October 1996. The same (AA), DON, and possibly NH4+ (Capone et al. 1994, parameters were measured in Trichodesmium IMSlOl Glibert & Bronk 1994, O'Neil et al. 1996). During isolated from coastal North Carolina and grown on oceanic blooms of Trichodesmium, dissolved N pools an N-depleted seawater medlum (Prufert-Bebout et al. can become elevated as a result of recent N input 1993). Colonies of Trichodesmium spp. were collected (Devassy 1987, Karl et al. 1992). Based on these obser- in the field using a 202 pm mesh plankton net towed at vations and the energy requirements for N2 fixation, Chesapeake Biological Laboratory (CBL). Light levels 15N uptake. Rates of N uptake were measured using in the incubators were about 55 to 65 pm01 quanta m-2 I5N tracer techniques as outlined in Glibert & Capone S-' PAR supplied by banks of cool white fluorescent (1993), using highly (>98%) enriched I5N substrates lighting. Cells were transferred using sterile tech- For field studies, colonies previously picked into GF/F niques under a laminar flow hood and maintained in filtered seawater were counted into polycarbonate exponential growth to prevent excess bacterial accu- incubation bottles containing 50 or 100 m1 GF/F fil- mulation. Non-Tnchodesmium bacterial abundance in tered, low-nutrient seawater (20 colonies per incuba- cultures was estimated as analyzed at the beginning and end of each set of 25 the ratio might be indicative of enzyme modification samples. Two reference samples were inserted after and the proportion of available enzyme that is biosyn- every 5th sample throughout the sample run to verify thetically active (Lee et al. 1988). instrument performance over the course of sample The HEPES extraction buffer and the reaction 'stop' runs. Reference samples were reproducible to within mix were prepared fresh daily. Reaction mixtures were 0.0001 atom %. prepared fresh just prior to initiating assays and GS activity. In field studies, 20 to 100 colonies of Tri- warmed to the assay temperature just prior to use. chodesmium spp. were rinsed and then counted into Transferase and biosynthetic assays were each set up incubation bottles containing fresh GF/F filtered sea- in triplicate for each sample. Assays were initiated by water. The contents of these bottles were then filtered adding cell homogenate to tubes containing the onto polycarbonate filters (3.0 to 8.0 pm pore size). Fil- warmed reaction mixture and the reaction proceeded ters were flash frozen in liquid N2 and stored at -80°C in a water bath or a heating block maintained at 30°C. until analyzed. In culture studies with Trichodesmium Background absorption was corrected for by stopping IMS101, the parent culture was gently mixed and a the reaction in 1 of the 3 replicate assay tubes immedi- known culture volume was gently filtered onto poly- ately after addition of the crude cell extract. Reaction carbonate filters, frozen and stored in liquid N2 until times were optimized for both the GS transferase and analyzed. CS biosynthetic assays by measuring activity over sev- GS activity was measured on crude cell extracts eral time courses using cell extract from freshly har- either immediately or after storage in liquid N2 for vested Trichodesmiurn spp. Reaction times were <2 wk. GS biosynthetic and transferase activities were selected from the portion of the curve in which enzyme measured using the y-glutamyltransferase and y-glu- activity increased linearly over time, 10 min for trans- tamylsynthetase assays of Stadtman et al. (1979) with ferase assay and 15 min for the biosynthetic assay. The the modifications described in Lee et al. (1988) after reaction mixtures were adjusted to pH 7.7 (transferase) extraction in HEPES buffer solution. The GS trans- and 7.5 (biosynthetic)using 10 N NaOH prior to initiat- ferase assay measures the amount of y-glutamylhy- ing the assays (Meeks pers. comm.). droxamate produced in the following reaction cat- The y-glutamylhydroxamate produced from the alyzed by GS: biosynthetic and transferase reactions was measured h04 using a Shimadzu UV-160 spectrophotometer and the gin + ADP + NH2OH Mn.. concentration determined relative to the standard y-glutamylhydroxamate + NH3 (1) curve associa.ted with each sample set. Replicate mea- This reaction does not occur in vivo but the y-glu- surements varied by less than 0.010 absorption units tamylhydroxamate produced can be measured spec- for transferase assays and 0.001 absorption units for trophotometrically and the reaction is analogous to the biosynthetic assays (usually lected during culture studies in the laboratory were N. An average colony was estimated to be 0.1 to 0.2 pg frozen and stored in liquid N, until analysis. Intracellu- N (Carpenter 1983a, Mulholland unpubl. results) lar pools were extracted in 0.3 N perchloric acid. The although this level varied with colony size. Cultures extract was neutralized and chloride precipitated with ranged from 0.7 to 1.0 pg N ml-l. N uptake measure- 2 M potassium carbonate as described by Senior ments were expressed in units of reciprocal time to (1,975).AA were analyzed by OPA fluorescence using avoid the problems of variable colony size and varia- reverse-phase high performance Liquid chromatogra- tions in total biomass among incubations and to allow phy (HPLC) as described by Flynn (1988) and Cowie & better comparisons with uptake rates measured previ- Hedges (1992). Analyses were performed on a Waters ously and uptake rates measured in cultures in which system with a refrigerated autosampler and fitted with colonies did not form. a Novapac column (150 X 3.9 mm, 4 pm, C-18 packing). Because the analytical error associated with the The mobile phase, supplied at a rate of 1.5 m1 rnin-l, measurement of AA, GS activity, N2 fixation by ace- was a gradient (solvent A: 0.025 M sodium acetate, 2 % tylene reduction and 15N uptake were negligible tetrahydrofuran, 0.05% Brij; solvent B: 100% meth- based on instrument performance and reproducibility anol). AA were resolved by modifying the gradient and between replicate assays, only that error associated pH of solvent A to achieve optimum separation. Indi- with differences between replicate samples is pre- vidual AA peaks were identified by comparison with sented in the results. Replicate measurements of acety- retention times from mixtures of pure AA standards. lene reduction and I5N uptake were made for most AA were quantified relative to a standard AA mix studies and errors are presented as standard devia- (Sigma A-2161) to which gln had been added. Stan- tions among sample replicates. Due to biomass limita- dards were run after every 5 samples. Distilled water tion in both natural and cultured populations and the blanks were run with each sample set and subtracted handling time necessary for sample collection and from the sample measurement. Neutralized perchloric preparation, replication was not possible in some of acid extraction blanks were run with each group of these experiments. samples extracted. Reproducibility of measurements to within 1 % was established by repeat injections. Stan- dard curves were run at the beginning and end of each RESULTS sample set (R2> 0.99). Only the glu and gln concentra- tions are reported here, akg was measured enzymati- Natural populations cally as described by Lowry et al. (1971) and Lowry & Passonneau (1972). N1 fixation and N uptake Inorganic nutrient analysis. Intracellular NH4+con- centrations were measured on a number of occasions Rates of NZ fixation, as estimated by acetylene by rupturing cells with a combination of heat and reduction, by Trichodesmium spp. varied among sam- osmotic shock (Thoresen et al. 1982). Interfilamental pling stations and sampling dates. The highest N nutrient concentrations were approximated by placing turnover rates attributed to NZ fixation, up to 0.4 % h-', 30 colonies into 30 m1 nutrient-free filtered seawater, (equal to 0.6 nmol N colony-' h-') were recorded at gently agitating them to disperse trichomes without mid-day (Table 1). The daily pattern of nitrogenase disrupting cells, and then collecting the resultant fil- activity for individual incubations and Tnchodesmium trate for nutrient analysis, as described in Capone et al. spp. populations was a bell-shaped curve centered (1994). Samples for measuring the ambient nutrient around mid-day as shown in previous studies (Table 1, and dissolved free amino acid (DFAA) concentrations Capone et al. 1990, 1994). While analytical precision in the GF/F filtrate used in each study were collected was high (see 'Methods'), replicate incubations could and measured immediately or stored frozen (-20°C) vary by up to 50%. This is consistent with variability until analysis. Nutrients (NH,', NO3-, urea) were mea- found in earlier studies (Carpenter & McCarthy 1975, sured colorimetrically using a Technicon autoanalyzer Carpenter & Price 1977, Capone 1990, 1994). Activity I1 (Friederich & Whitledge 1972) equipped with an decreased over the course of the afternoon and was Alpchem autosampler and FASPac software. DFAA absent by evening. N2 fixation was not detected at were measured by HPLC as described above. Concen- night. trations were determined by comparison with standard N uptake (NH,', NO3-, urea, glu and a mixed AA curves (R2> 0.99). Repeat measurements were made substrate) was detected in Trichodesmlum spp. col- from the same sample to verify reproducibility of mea- lected from low nutrient areas of the North Atlantic surements to within about 2 %. Ocean and the Eastern Caribbean Sea and N uptake Field measurements were normalized per colony rates varied among populations, between sample and culture measurements were normalized per unit replicates, and relative to calculated rates of NZ fixa- 1owu I> A~~ensn)u16 jo suogelluasuoa '(~'37,,6!,1) [enlse ay1 IsaIjal IOU Aeu~palelnqes sanleh sxMO~ uogexg ZN jo sale1 ql!~1a11e~ed U! pasealsur aql OS pue ~rlo,&ueql ssa1 suogequasuos le papalap L11elaua6 suo~lerluasuosu16 UI sasealsu1 .alom payen lou SPM -CON JO ayeldn '(I ayeldn -CON 10j AI! aurg awes aql le palsal~ossa~dures 6uoure uoqeuelz -3edes IsaMoI aql pue ayeldn +PHN JOJ (X9w~)Al1~ede3 '% I> dq paueh shesse alesqdal q6noql1~.'(g'~z 'By) 1saq61q aql peq satuolos .dds urnrursapoyJ,uL ,saleqs uooulalje aql U! paugsap pup 6u1uiour aw U! AI -qns pue suo~lels6uoure pauea sluelsuos xlau~)~ -lea lsaqB!q alaM suoqequa3uos 6yn ~e~nl~ase~lu~ ,hp-p!tu punole A~uosale1 lenr~xeurle ZN x!j spaleql MOI~anz uaqM (lalea16 awl 6u11qnop aql PUP) JaMoI aq Leu uorlexg " 01 anp lanoulnl N -pop 01 szlnoqe JO sauq Gugqnop p1a1d pInoM s1y~'LPM -qled uojlezl[!ln N sq~Aq A[rep pap~~o~daq pp02 ssem .suralsLs [elnieu U! luel~odur! -o!q N Xuo~os[et01 aql jo % p jo unwrxem e 'uo~lex~jZN aq Lew spunodmos asaql jo ayeldn 'sIIas aql punole jo pouad q 01 e uo paseg .p s.8 lnoqe JO aurq lalzouinl eale aql U!: suo!lr?qua2uoJ luaulnu q61q osle ale alaql paseq N e salnl!lsuos srq~'UO!IPXIJ ZN 01 palnq!lllp asu~spue saleqsqns N z asaql loj dl!u!jje pup Al!sedes SPM %, C'Zlnoqe pue 'pouad q pz e 6ur~np1aAo paulnl ayeldn q61q e aheq sIlas asnesag .palnseaw a1aM seM N Ilas aql JO %c-[lnoqe JO lelol e a6ela~euo ~rlSIC ol dn jo suo~le~luasuosea1n aI!qM mrl IIZ .aIqelsalapun 10 MOI alaM ayeldn CON^,30 sales se q6ry se alaM +PHN jo suo~lequasuosIeluaruelrj 'mu 0s > a~a~QV jo suo~lelluasuos lua!qurv .pal>alap -laluI '(ca~qe~) suo!lelluasuos umnlos laleM lualqme slienz1.e Inq laMo1 alaM salw aye~dn QV paxy 01 aa!lelal paleAaIa A-[-[eiaua6alaM sa!uoIos yl!~pale pue 1-113',-q y, 11.0lnoqe 01 olaz uo~jpa6uel pue -!sosse earn pue ,~HNjo suogelluasuos le[n[[asellxg sa!pn.ls 6uowe alqerielz alaM ayeldn ealn 30 sale8 .sa!uoIos 30 uo!le6al66es!p a(lua6 Aq .dds rrmrursaporp 'suo~l~pp~wrt ~0.0 dq pale.[nur!ls uaaq aAeq -.uJ.30 suogolndod auos ur saseds IeluaweI!jJalu! Aeur ayeldn N,, 30 sale1 'uo!lsalap 30 sl!w!1 aql Mopq u1 palnseaur alaM suo!lequasuos +~HNpue ealn 10 le alaM *b~~jo suorlequasuos lua!qwe asnesag .SUOIl .Xep aql 6u!mp palnseaur araM ',-q %8'1 ol dn 'sale1 -elnsIes lno u1 paleunlsalapun aq Aeur san-(eA "S + 'X ayeldn +PHN 1saq61q aqL '(1 alqe~)uo!lex~j ZN ou SPM OS pue mri O&le palelnles IOU SPM ayeldn ealn .(I -6!g) alaql uaqM lq61u aql6uynp papaasold pue hpaql jo salellsqns N asaql ~ojAl~urjje q61q e 6u1les!pu1 'ayeldn lsow lnoq6noiql UOIIPXIJ 2~ JO sale1 papaasxa ayejdn vv pue n16 'TP~~~ojpaleInsIes alaM sanleA mu^y61y +PHN 'salellsqns N lay10 103pa.rnseam asoyl ueyl pue "S + MOI AI~AIIPI~~,alellsqns s!ql 103 dl!u!jje lay61y r(11elaua6 aiaM ayeldn +P~~s,30 sale8 .uoq (&ro,o)11.0 aN OO:IZ ~6/90/z1 (~0'0)800 aN CS161 96/P0/6 1 (900'0) 50'0 100.0) S1O.O (1000'0) t000.0 (1000.0) P000'0 (100'0) PO-0 aN OS:81 96/01/8Z (HN) S9.O aN 00:91 96/O 1/EI (~0.0)11'0 EZ:S1 P6/90/ZI (90'0) 61.0 (~0.0)QPO'O 01:St 96/P0/61 iz0.01 P0.0 (96'0) P8'1 (~00'0)CZO'O 00:SI 96/L 1/90 (CP'O) 8s 0 90:PT 96/PO/C1 (HN) ZC.0 (OP'O)LC'O (8z.0) 010 (~0'0)OP'O SP:E1 96/01/91 (900.0) Z10'0 (900'0) PO'O QN (LZ'O)PP0 S1:11 96/0 1/61 (zoo.o)r 10.0 (100.0) s1o.o (coo.o)zoo-o (~ooo.o)cooo.o (100-0) 900 (01.o) pc.0 OT:~I 96/0t/~z aN (HN) to-0 SP:OI 96/01/LI (10'0) COO (~0.0)LZ-0 (Po'o) CZ'O 8P160 P6/90/Z1 (HN) 60.0 (HN) CO'O (HN) CPOO'O (HN) L90-0 (~00'0)10'0 00:tO 96/01/0Z (80.0) 60'0 aN 0O:PO P6/9O/ZI (,-q %l (,-v %l (,-v%o) (,-q %) (,-q %I (,-q ay eldn a yeldn ayqdn ayeldn aye~do uogex~j NI (~A/o~/P) VV n19 ea~n -CON +P~~x~ aWk a3ea saldues ales~~da~ou :g~ .palsa)ap SPM uo!leTj ZN 10 aqeldn N ou :aN.sasaqluaied U! paliodai alp suo!le!*ap piepuels ',.Auolos N le 1.0 perl Auo103 a6era~eaql .A~an!lsadsai 'NU 001> pue o[> aiam suorleiluasuos (vv)p~se ou~ure pue (n13) a)ew -ew6 lua!qure PUP ~0.0)uorlsalap jo sl!m!l aql MoIaq 10 le alaM earn pue '?ON '+PHN jo suogellumuos jua!qury ,wtl ~0.0 aJalw saleilsqns N~,jo suo!l!ppe lax11 'ueaso s!luellv lsaMqlloN lesldoilqns pue le31dol1 aql worj p~llsallosaram sa~dwes 1IV 'alW Aq '-dds ~n!cuSapOrlJ!JLU! (,.q N uo!lex!j i~ pue (,-q N %) ayeldn N jo saler palnseaw jo hewtuns -1 alqel Mulholland & Capone: Nitrogen metabolism in Trichodesmiurn spp. 3 9 NO; C D 2 - Urea Glutamate K. = 3.1 (1.29) PM 0.8 - 0.4 -- 0 l 0 10 20 30 40 0 10 20 30 40 Concentration (PM) Fig. 1. Saturation curves for combined N sources: (A) NH,'. (B) NO3-, (C) urea, and (D)glutamate in natural populations of Trj- chodesmium spp. from the tropical North Atlantic Ocean. Value for + S,,(standard deviation) and G',,,, indicated in each panel. All incubations were done during the morning after sunrise. ND:value could not be determined colony-') were always lower than concentrations of glu Intracellular pools of glu and gln and the ratio (<5nmol colony-'). As previously reported (Capone et between these pools varied depending on the depth al. 1994), the peak in intracellular glu concentrations from which Trichodesmium colonies were collected. was slightly later in the day than that for gln The glu and gln concentrations were highest in (Fig. 2C,D). There was an apparent die1 pattern in colonies collected from just below the surface (5 m) metabolite ratios in Tnchodesmium spp. The gln/akg and then decreased with depth (Fig. 3A). The gln/glu was highest before or at mid-day and lowest during the ratio was highest at the surface and decreased with night (Fig. 2E,F). The ratio ranged from about 0.2 to 2. depth (Fig. 3B).The highest gln/glu and glnlakg ratios The highest gln/glu ratios were measured before or measured were about 0.7 and 7, respectively, in sam- about mid-day (range 0.1 to 0.6) (Fig. 2G,H). ples collected from near surface waters. The ratios Intracellular NH,' pools ranged from 209 to 1822 pM rapidly decreased to between 0.2 and 0.4 for gln/glu (Table 3), similar to the ranges of values reported for and <2 for the gln/akg for samples collected deeper in glu and gln. the euphotic zone (Fig. 3C). In experiments in which Trichodesmium spp. colonies were collected from 15 m and then incubated Table 2. Concentrations of nutrients in interfilamental areas on deck at various simulated light levels, there were no of Trichodesmium spp. colonies from the North Atlantic Ocean. Calculations were based on an average colony size of consistent patterns in intracellular glu and gln concen- 5 by 0.5 mm. Ten percent of the total cylindrical volume was trations with respect to light level (data not shown). interfilamental spaces (Capone et al. 1994). Standard devia- Intracellular pools of akg were lowest at the higher tions from 3 replicate analyses are reported in parentheses. light levels and, therefore, the gln/akg ratio was ND: nutrient was not detected. The detection limit was 0.03 pM slightly higher in the high light treatment but did not change much compared to the lower light treatments. Date NH,' conc. (PM) Urea conc. (PM) (d/mo/yr) Ambient Interfilamental Arnblent Interfilamental GS activity 31/05/94 ND ND ND 182 (4.9) GS transferase activity was, on average, between 8 and 16 nmol y-glutamylhydroxamate pg-' protein h-', more than 20 times higher than GS biosynthetic activ- 4 0 Mar Ecol Prog Ser 188: 33-49. 1999 lax, I ohm 7 0 400 80012001m20002400 0 403 80012001600m2400 Time (h) Fig. 2. IntraceLlular concentrations of (A,B)a-ketoglutarate, (C,D)glutamate and glutamine; intracellular ratios of (E,F)gln/u.kg and [G,H) gln/glu in natural populations of Trjchodesmium spp (tufts) from (A,C,E,G)the tropical North Atlantic Ocean and [B,D,F,H) the Caribbean Sea Error bars are standard deviations calculated from 3 Trichodesmium spp. samples. Bars on the x-axis indicate the dark period Curves were drawn in Excel" using a 4th order polynomial regression Date Colony Time NH,+ NH,' (d/mo/yr) form conc. conc. (nmol colony-') [PM) 31/05/94 Tufts and puffs 1000 0.58 209 05/06/94' Tufts 1000 1.47 868 05/06/94" Puffs 1000 2.67 494 Table 3. Intracellular NH,' pools in Trichodesmium spp. 28/05/94 Tufts and puffs 1330 0.83 301 collected from the tropical North Atlantic Ocean. All 06/06/94d Tufts 1330 0.77 454 measurements are reported. Rephcate samples were not 06/06/94" Puffs 1339 0.57 1050 collected on these days. Tufts represent fusiform bundles of 3 1/05/94 Tufts 1500 3 42 809 trichomes and puffs are a radial arrangement of trichomes. 31/05/94 Puffs 1500 2 46 1820 Intracellular concentrations were calculated based on an average of 100 cells trichome-l (Marumo 1975, McCarthy & these days the average colony size was estimated to Carpenter 1979) and cell volumes of 540 pm3 for spherical be about 100 trichomes colony-'. On the other days, the colonies (puffs) and 1690 pm3 for fusiform colonles (tufts) average colony was comprised of about 250 trichomes (Carpenter 1983a) Mulholland & Capone: Nitrogen metabolism in Tnchodesmium spp. 4 1 Concentration (pmol col') Cultured populations 0 loo0 2000 3000 4000 Biomass A The average N biomass of cultures of Tricho- desrniurn IMSlOl used in these experiments was 121 pMol N 1-l. At this density, cells were in expo- nential phase growth. N, fixation Ratio Assays for N2 fixation were initiated at 4 points dur- 0 ing the light period and twice during the dark and monitored over time. In cultures of Trichodesmium IMS101, the highest average rates of N2 fixation GlnlGlu (1.05% h-') were measured during the middle of the light period (Table 4). These rates exceeded the rates of N2 fixation measured in natural populations of Tri- chodesmium spp. colonies. Activity increased after the onset of the light period and decreased during the lat- ter half of the light period (Table 4).N2 fixation was not detected during the dark period. Integrating over the day and assuming maximal rates of N2 fixation 250 throughout the 10 h light period, we estimate that Ratio there was a turnover of 11 % of the cell N per day due 0 2 4 6 8 to N2 fixation. This translates to an N2 fixation-based N 0 doubling time of about 9 d. 50 Nitrogen uptake 100 Depth Prior to the addition of Tnchodesmium, NH4+, glu (m1 and gln were not measurable in the seawater 150 medium. NH4+ accumulated in the culture medium during growth of Trichodesmium IMSlOl, with con- 200 centrations ranging between 0.3 to 1.8 FM over the diel cycle (Table 4). Additions of I5NH4+were < l0 % of the ambient NH,+ concentration in the medium. Fig. 3. Intracellular (A) glu and gln concentrations, (B)gln/glu Additions of I5N-labeled glu represented 100% or ratios, and (C) gln/w.kg ratios in Trichodesmium spp. collected more of ambient concentrations. Average rates of at a single station from different depths on the same day in NH,+ uptake were highest (average of 0.68% h-') just the Caribbean Sea after the onset of the light period and then were lower at mid-day (0.2 to 0.3 % h-') when N2 fixation rates were high (Table 4). Uptake of NH,' repre- ity (4 to 6 nmol y-glutamylhydroxamate pg-' protein sented about 20 to 40% of the N2 fixation rate at mid- h-'). Levels of activity were highest after mid-day and day. Rates of NH,' uptake were greater than the lower during the early morning. The ratio between GS rates of N2 fixation except around mid-day when N1 transferase and GS biosynthetic activity varied only fixation rates were highest. In the afternoon, NH,' slightly during the course of the day, averaging uptake rates were lower than in the morning. Inte- between about 18 and 25. There was a slight increase grated over a day, NH,' uptake accounted for 10 to in the transferase/biosynthetic ratio in the afternoon 16.3% of the total cell N per day. There was a strong but there was no significant trend. Few measurements positive relationship (R2= 0.90) between the rate of were made during the dark period. As a result, dis- NH,+ uptake and the NH,' concentration measured cerning diel changes in activity is difficult. in the culture medium (Fig. 4). Mar Ecol Prog Ser 188: 33-49, 1999 Table 4. Summary of N uptake and Nz fixation rates by Trichodesmium IMSlOl from experiments conducted on 12/06/96 using 0.03 PM additions of "NH,' or 'SN-glutamate. Rates are expressed as turnover rates of 9/0cell N h-'. The standard deviations from 2 replicate incubations are reported in parentheses. ND: rates were not detected Time N2fixation NH4* NHI* Glu Glu (h) ('Y"h l) uptake in medium uptake in medium (% h-1) (PM) ('Wh-') (1.w Glu uptake was low, but detectable, throughout the GS activity diel cycle (Table 4).Rates were about 0.03%D h-', yield- ing an average daily N turnover due to glu of 0.72% Both GS transferase and GS biosynthetic activity d-' Glu concentrations in the culture medium ranged decreased after the period of high N, fixation in the from about 0.02 to 0.06 PM. Therefore, the 0.03 pM 15N afternoon (Fig. 6A). Similarly, the ratio of GS trans- tracer additions represented 50 to 100% enrichments ferase/biosynthetic activity decreased from about 26 to of the ambient glu concentration (Table 4). These high 20 during the period when rates of N2 fixation were additions may have stimulated uptake and therefore high (Fig. 6B) Late in the light period the ratio the results should be considered as upper estimates. increased to val-ues over 30 and remained about 30 The uptake of glu did not correlate with the concentra- during the dark period. tion of glu in the medium. Intracellular metabolite pools Intracellular pools of glu and gln increased during the light period in TrichodesmiumIMSlOl growing on medium without added N substrates (Fig. 5A). The highest concentrations were measured during the period of high N, fixation. Intracellular concentrations of glu were more than 10-fold higher than gln concen- trations during most of the day. There was a strong diel pattern in the gln/glu ratio in Tr~chodesmiumIMSlOl 0 400 800 1200 1600 2000 2400 (Fig. 5B).The gln/glu ratio rose to a peak of about 0.3 Time (h) during and just after the period of highest N, fixation. However, during the dark period, when there was no N2 fixation, ratios decreased to about 0.1 d InlGlu Ratio 0 400 800 1200 1600 2000 2400 Time (h) Fig. 5. Intracellular pools of (A) glu and gln and (B)the gln/glu ratio relative to the pattern of N2 fixation measured (actual values shown In Table 41 in Trichodesmium IMStOl srown- on seawater medium depletedin combined N substrates. Bars on Fig. 4. NH,- uptake relative to concentrations of NH,' mea- the x-axis indicate the dark period (darkness from 22:30 to sured in the culture medium 08:30 h daily) Mulholland & Capone: Nitrogen metabolism in Trichodesmium spp. 43 A tions growing on medium without added N (Table 5). GSTransferase An induction period for NO3- transport systems and NO3- assimilatory pathways or some minimum concen- tration may be necessary for NO3-~til1zati0nby natural and cultured populations (Flores & Herrero 1994, Mul- holland et al. 1999). In general, the rates of urea uptake measured in this study (Table 5) were much higher than those measured by Carpenter & McCarthy (1975). However, these rates are consistent with rates reported by Saino (1977). We also measured high concentrations of urea within interfilamental spaces of colonies of Tncho- Time (h) desmium, suggesting that urea may be produced and taken up within the colonial assemblage. The source of this urea is not known. It is possible that urea is produced by grazers Living in association with colonies. Bacterial production of urea has been mea- sured in sediments (Pedersen et al. 1993) and seawa- ter (Slawyk et al. 1990) but its production in associa- tion with Trichodesmium spp. colonies has not been measured. Trichodesmium spp, have the highest capacities for NH,' and AA uptake. In natural populations of Tri- chodesmiurn we measured NH,' uptake rates of 0.05 to 1.84 % h-'. These rates are higher than those measured 0 400 800 1200 1600 2000 2400 by Carpenter & McCarthy (1975) but similar to the Time (h) rates reported by Saino (1977) and Goering et al. Fig. 6. (A) Glutamine synthetase (GS) transferase and biosyn- (1966). Bacterial utilization and regeneration of N sub- thetic activity and (B) the GS transferase/biosynthetic activity strates, particularly NH4+ and AA within incubation ratio relative to the pattern of N, fixation measured (see bottles can cause variability in measured rates of N Table 5 for data) in T~chodesmi~mIMSlOl grown on sea- uptake (Kirchman et al. 1989). While bacteria can con- water medium depleted in combined N substrates. Bars on the x-axis lndicate the dark period (darkness from 22:30 to sume NH,', Kirchman et al. (1989) demonstrated that 08:30 h daily) high DON (specifically as AA) concentrations stimu- late net regeneration of NH,' by bacteria. Enrichment of interfilamental spaces with AA (Carpenter et al. DISCUSSION 1992, Capone et al. 1994) may result in a net produc- tion of NH,' from AA in Trichodesmium communities. N utilization Bacteria occur in association with Trichodesmium colonies and are both directly attached to filaments Rates of N2 fixation vary widely among populations and in loose association with colonies (Paerl et al. of Trichodesmium from different oceanic regions 1989). Carpenter & Price (1977) estimated that Table 5. Rates of N uptake by Trichodesmiumspp. reported in the literature and those measured in these studies. Glu- glutamate, Gln: glutamine. AA: amino acids, ND: not detected Substrate Location Addition N uptake Uptake rate Uptake rate Source (I.IM) (l' h) (pg N cell-' h-') (pg N colony-' h-') NH4' W Sargasso Sea 0.13-20.4 0.0006-0.018' 0.02-0.59 0.6-17.7~ Carpenter & McCarthy (1975) W Sargasso Sea 0.067 0.005-0.014 Carpenter & McCarthy (1975) W Sargasso Sea 14 0.0042 .0.0048' 0.14-0.16 4.2-4 a'.' Carpenter & McCarthy (1975) N Atlantic 2 0.005- 0.08 Goering et al. (1966) N Atlantic 2 0.51 (Bloom) Goering et al. (1966) NW Pacific 0.03 0.01 Saino (1977) NW Pacific 0.05 0.02 Saino (1977) NW Pacific 0.1-1 .O 0.035-0.23 Saino (1977) NW Pacif~c 2.0-10 0.03-0.21 Saino (1977) NW Paciflc 2 0 2.0 Salno & Hattori (1978) N Atlantic 0.03 0.05-2.0 Present study No3- W Sargasso Sea 0.13-20.4 -0 -0 Carpenter & McCarthy (1975) W Sargasso Sea 14 0.00012-0.0003' 0.004-0.01 Carpenter & McCarthy (1975) N Atlantic 2 0-0.02 Goering et al. (1966) N Atlant~c 2 0.175 (Bloom) Goerlng et al. (l966) NW Pacific 10 0.0026-0.0028 Saino (1977) N Atlantic 0.03 ND-0.0005 Present study Urea W Sargasso Sea 0.13-20.4 -0 -0 Carpenter & McCarthy (1975) W Sargasso Sea 14 0.00054-0.0006C 0.018-0.02 Carpenter & McCarthy (1975) NW Pacif~c 10 0.0494-0 0534 Saino (1977) N Atlantic 0.03 ND-0 2 Present study Glu NW Pacific 10 0.0085-0.0093 Saino (1977) Caribbean 0.07 < 0.2 Capone et al. (1994)~ Caribbean 0.001 0.0018-0.0053' 1.8-5.3 (am) Carpenter et al. (1992)' Caribbean 0.001 0.025' 25 (mid-day) Carpenter et al. (1992)' Present study N Atlantic 0.03 U.0081-0.69 2 8-952 Gln Caribbean 0.001 0.0001-0.00007' 0.1-0.07 (am) Carpenter et al. (1992)e Caribbean 0.001 0.0025-0.005' 2.5-5 (mid-day) Carpenter et al. (1992)e Glycine N Atlantic 2 0.02 Goering et al. (1966) AA N Atlantic 0.03 0.01-0.023' 2 3-410 Present study 'Mostly non-detectable bCalculated based on Carpenter & McCarthy's estimate of 30000 cells colony-' CCalculated based on estimate of 100 ng-at N colony-' dUsing I4C-labeled glu 'Using 'H-labeled glu and gln chodesrnium. This may reflect the lower abundance of N metabolism the microbes necessary for regenerating NH4+from glu in these populations. Intracellular N and C dynamics and the availability of We found that in natural populations of Tricho- substrates for NH4+assimilation have been implicated desmium spp., K, + S, values for NH,' and glu were as important factors affecting the regulation of N2 fixa- consistent with those reported previously (Table 6). tion and N metabolism in a variety of bacteria and Our V,,, estimates for NH4+uptake were substantially cyanobacterla (Guerrero & Lara 1987, Flores & Herrero higher than those previously reported. In cultures of 1994). Gln/glu ratios have been utilized as an index of Trichodesmium NIBB1067, apparent V,,, decreased cellular C:N status and N limitation in a variety of as incubation length increased, suggesting that recy- microalgae (Flynn et al. 1989, Flynn & Gallon 1990). cling of nutrients, and consequent isotope dilution, Flynn et al. (1989) suggested that phytoplankton cells may occur within the incubation bottles during longer with intracellular gln/glu ratios >0.5 are N-replete incubations (Mulholland et al. 1999). Because our while those with ratios of <0.2 are N-depleted. Apply- incubations were short (1 or 2 h), isotope dilution was ing these criteria, Tnchodesmium spp. cells are rarely minimal during these experiments. N-depleted. We observed gln/glu ratios of 0.6 during Mulholland & Capone: Nitrogen metabolism in Trichodesmium spp. 4 J Table 6. Values of [PM)and V,,, (% N h-') reported for various N sources and therefore had the potential to assimi- those measured in these studies late enough N to turn over their cell N at least 50 times d-l, suggesting that N assimilation does not limit the Substrate K ~"MX Location Source (PM) (% h-') rate of N utilization by cells, even when NZ fixation rates are maximal. NH4+ 6.74 0.177' W Sargasso Sea Carpenter & McCarthy (1975) Some investigators have reported 0.3 to 12 0.045 to 0.25 NW Pacific Saino (1977) that only a subset ( N budgets productivity estimates based on N2 fixation versus C fixation in natural populations of Trichodesmium. In Based on total measured N utilization (NZfixation the Caribbean Sea, Carpenter & Price (1977) estimated plus NH4+and glu uptake), an N-based turnover time that Trichodesrnium spp. represented 20% of the car- of about 3 to 5 d was calculated for Trichodesmium bon productivity but only 8% of the system's N IMSlOl during exponential phase growth. When only demand was supplied by N, fixation. In Tricho- N2 fixation was considered, 9 d were required for a desmium IMS101 cultures, we calculated that C and N doubling of the culture biomass. At similar biomass turnover were nearly balanced relative to the reported levels, Trichodesmium NIBB1067 doubles in 8 d and is C:N ratios (Carpenter 1983a. Carpenter & Romans in late exponential growth phase (Mulholland et al. 1991) only when we consider both N2 fixation and 1999). NH,+ uptake as components of total N turnover. The N Independent of absolute growth demands, we calcu- demand calculated from short-term C productivity lated a daily N utilization budget for Trichodesmium measurements may overestimate N requirements for IMSlOl by integrating the relative contribution of 3 net growth. In cultures of Trichodesmium grown on N sources (N2,NH4+, and glu) to the total measured N medium without added N sources, N2 fixation was a turnover over a diel cycle (Fig. 7). Of the total daily good indicator of net growth N utilization, 72 % was attributed to NH4+uptake, 23 % Observations of high N turnover rates relative to was due to N, fixation and 4% was from glu uptake. rates of N2 fixation combined with low 6I5N signatures Because our cultures of Trichodesmium were closed may be reconciled if there is a tight coupling between systems to which no combined N had been added, all Nz fixation, NH4' release or N regeneration and uptake of the N used to support net growth must ultimately be of the low 8I5NH4+derived from recently fixed NZ. At derived from N2 fixation. present, there are no determinations of the 615N signa- Based on C:N ratios observed in colonies of Tricho- ture of the NH4+pools in these systems to confirm this desmiurn (Carpenter 1983a), the stoichiometry of C speculation. Zooplankton grazing on Trichodesmium and N2 fixation appear to be out of balance. For and other N2-fixing organisms may contribute to an instance, Capone et al. (1998) calculated that N, fixa- isotopically light NH,' pool in the upper water column. tion provided < 20 % of the daily N demand necessary Checkley & Miller (1989) have observed that oceanic to balance the observed C utilization by Trichodes- zooplankton excrete NH4+ that is isotopically lighter nxum populations within an Indian Ocean bloom. than their body values. However, the low 6I5N of these populations suggested Carpenter et al. (1999) reported that the relative that N2 fixation was still the primary N source for these contribution of NH4+to the total N nutrition increased populations. Other imbalances have been observed in over time in a bloom of an Nz-fixing symbiosis, Hemi- aulus/Richelia. However, the population retained a light 6I5N signature. We speculate that Trichodesmium may exhibit a similar pattern whereby populations alleviate N limitation initially by fixing N2. Subse- quently, newly fixed N may be recycled as NH4+and DON. These N sources may be available for uptake by cells to support growth. Similarly, the relative propor- tions of N uptake and N2 fixation to total N turnover may change over a growth cycle in cultures of Tn- chodesmium. Experiments are underway to test this hypothesis. Community N dynamics A high affinity for NH,', coupled with the positive Time (h) correlation between NH4+ concentration and NH,+ uptake in cultures of Trichodesrnium IMS101, suggest Fig. 7. Total daily N turnover by Trichodesmium IMSlOl that NH4+release and uptake may be tightly coupled growing on medium depleted in combined N substrates within Trichodesrnium spp. communities. Tight cou- (upper panel). Percent contribution of glu, NH,' and N2fixa- tion to the daily N turnover. N2 fixation N and glu and NH,' pling between N, fixation and NH,+ uptake and a uptake rates were measured over a diel cycle and these rates capacity for concomitant N2 fixation and N uptake may were integrated over a 24 h day be an important adaptation whereby Trichodesmium Mulholland & Capone: Nitrogen metaboLism in Trichodesrnium spp. 47 spp. can support growth and maintain their biomass in centrations increased to almost 2.0 pM in cultures extremely oligotrophic environments. In closed culture grown on medium without added N substrates. Saino systems, NH,' is retained in the system and may accu- (1977) also observed release of NH,' from Tricho- mulate in the culture medium for utilization by the cul- desmium colonies over time. tured organism. By contrast, in natural systems that are open, NH,' and DON produced by and released from cells or regenerated within colonial associations may Conclusions diffuse away from cells and become part of the am- bient nutrient pool available for utilization by all The previously reported low rates of combined N org-anisms in the community. Sometimes, high con- uptake by Tnchodesmium (Carpenter & McCarthy centrations of NH,' and glu were measured in the in- 1975, Glibert & Banahan 1988) and their low 6I5N sig- terfilamental areas of Trichodesmium colonies col- nature (Carpenter et al. 1997) have led to the general lected from natural populations (Capone et al. 1994, conclusion that NZ fixation is the primary mode of N Table 2). This suggests that colonies may retain nutri- acquisition by these species (Carpenter et al. 1997). ents in microzones thereby preventing their diffusion Our results indicate that, while NZ fixation may meet away from cells. the bulk of the cellular N demand for net growth, Tri- There are a variety of potential NH,' sources with- chodesmium spp. assemblages can concurrently utilize in the Trichodesmium consortia. AA and DON are combined N (mainly NH,' and DON) and fix NZat high released by Trichodesmium during growth (Capone rates. If only 10 or 15 % of Trichodesmium cells are able et al. 1994, Glibert & Bronk 1994). AA oxidase activ- to fix NZ, the release of fixed NZ and its subsequent ity associated with Trichodesmium spp, can liberate uptake may be an important mechanism for transport- NH,' from these compounds (up to 20 pm01 N ing N among cells that are fixing NZ and those that are colony-' h-') (Mulholland et al. 1998). Bacteria can not. also regenerate NH,' in environments rich m N-rich dissolved organic material (Kirchman et al. 1989). Nausch (1996) estimated that 0.32 to 15 nmol N Acknowledgements. We thank J. Burns, C. Shoemaker, G. colony-' h-' was released by peptidase and P-glu- Yellin, and S. Jefferson for their assistance at sea and in the lab. Ha1 Schreier and Jack Meeks provided helpful insights cosamidase activlty associated with bacteria residing regarding GS assays and we thank them. We thank Lee among Trichodesmium spp. colonies. Zooplankton Bebout for providing us with Trichodesmium IMSlOl and grazing can also result in rapid NH4+ recycling. helping us to keep it alive. We also benefited greatly from the O'Neil et al. (1996) reported NH4' release rates of support, cooperation, and companionship of the officers and crew of the RV 'Seward Johnson' as well as the other mem- 2 nmol N copepod-' h-' from Macrosetella gracilis bers of the shipboard scientific party. D.G.C. was supported grazing on Trichodesmium spp. They estimated that by NSF grants. 33 to 45% of a T. thiebautii colony and more than 100% of the N, fixed d-I could be consumed by graz- ing each day. The N content of a single colony is on LITERATURE CITED average about 100 ng N colony-'. Trichodesmiurn can also contain cyanophages (Ohki 1999). Viral cell lysis Bergman B (1999) Distribution of nitrogenase in the marine causes cells to burst, releasing the contents of the cell non-heterocystous cyanobacterium Trichodesmium. In: Charpy L, Larkum AWD (eds) Marine cyanobacteria. Bul- (including N metabolites and organic compounds) letin de I'lnstitut Oceanographique, Monaco, Special Issue into the environment. Our analysis of intracellular No. 19 (in press) NH,' (0.57 to 3.42 nmol colony-'), glu (1 to 5 nmol Bergman B, Lindblad P, Pettersson A, Renstrom E, Tiberg E colony-'), and gln (0.2 to 1 nmol colony-') concentra- (1985) Immuno-gold localization of glutamine synthetase in a nitrogen-fixing cyanobacterium (Anabaena cylin- tions would suggest that 3 to 9.5 nmol colony-' of drica). Planta 166:329-334 these combined N sources could be released when Bronk DA, Glibert PM, Ward BB (1994) Nitrogen uptake, dis- cells lyse. Based on the high rates of N regeneration solved organic nitrogen release, and new production. Sci- estimated from bacterial processes and grazing asso- ence 265:1843-1846 ciated with Trichodesmium spp. colonies, sufficient Capone DG (1993) Determination of nitrogenase activity in aquatic samples using the acetylene reduction procedure. NH,' substrates are available to support the rates of In: Kemp PF, Sherr BF, Sherr EB, Cole JJ (eds) Handbook NH4+uptake reported here (up to 55 pm01 N colony-' of methods in aquatic microbial ecology. Lewis Publishers, h-'), New York, p 621-631 In contrast to results reported by Glibert & Banahan Capone DG, O'Neil JM, Zehr J, Carpenter EJ (1990) Bas~sfor die1 variation in nitrogenase activity in the marine plank- (1988), our results also support a substantive direct tonic cyanobacterium Trichodesrnium thiebautii. Appl release of NH,' by Trichodesmium despite excess Environ Microbiol 56:3532-3536 biosynthetic capacity via GS. In this study, NH4+con- Capone DG. Ferrier MD, Carpenter EJ (1994) Amino acid 4 8 Mar Ecol Prog Ser 188: 33-49, 1999 cycl~ngin colonies of the planktonic marine cyanobac- Flynn KJ (1990) The determination of nitrogen status in terlum, Trichodesmium thiebautii. Appl Environ Microblol microalgae. Mar Ecol Prog Ser 61:297-307 60:3989-3995 Flynn KJ, Gallon JR (1990) Changes in intracellular ana Capone DG, Zehr JP, Paerl HW, Bergman B, Carpenter EJ extracellular a-amino acids in Gloeothece during NZ-fixa- (1997) Trichodesmium, a globally significant marine tion and following addition of ammonium. Arch Microbiol cyanobacterium. Science 276:1221-1229 153:574-579 Capone DG, Subramaniam A, Montoya JP, Voss M, Humborg Flynn KJ, Dickson DMJ, Al-Amoudi OA (1989) The ratio of C, Johansen AM, S~efertRL, Carpenter E J (1998) An glutam~ne.glutan~atein microalgae: a biomarker for N- extensive bloon~ of the N2-fixing cyanobacterium Tn- status suitable for use at natural cell densities J Plankton chodesnlium erythraeum in the central Arabian Sea. Mar Res 11:165-170 Ecol Prog Ser 172:281-292 Fredriksson C, Bergman B (1995) Nitrogenase quantity varies Carpenter EJ (1983a) Physiology and ecology of marine diurnally in a subset of cells within colonies of the non- planktonic Oscillatoria (Trichodesmium) Mar Biol Lett heterocystous cyanobacteria Trichodesmium spp. Micro- 4 69-85 biology 141.2471-2478 Carpenter EJ (198313) Nitrogen fixation by marine Oscillatoria Fredriksson C, Bergman B (1997) Ultrastructural characterisa- (Trichodesmium) in the world's oceans. In: Carpenter EJ, tion of cells specialised for nitrogen fixation in a non-hete- Capone DG (eds) Nitrogen in the marine environment. rocystous cyanobacterium. Trichodesrnium spp. Proto- Academic Press. New York, p 65-103 plasma 197~76-86 Carpenter EJ, McCarthy JJ (1975) Nltrogen fixation and Friederich GO. Whitledge TE (1972) Autoanalyzer proce- uptake of combined nitrogenous nutrients by Oscillatoria dures for nutrients. In: Pavlou SP (ed) Phytoplankton (Tn~chodesmium) thiebautii in the western Sargasso Sea. growth dynamics, Vol 1 University of Washington Special Limnol Oceanogr 20 389-401 Report 52, Seattle, WA Carpenter EJ, Price CC (1976) Manne Oscillatoria (Tn- Glibert PM, Banahan S (1988) Uptake of combined nitrogen chodesmium): explanation for aerobic nitrogen fixation sources by Trichodesmium and pelagic microplankton in without heterocysts. Science 191:1278-1280 the Caribbean Sea: comparative uptake capacity and Carpenter EJ, Pnce CC (1977) Nitrogen fixation, distribution, nutritional status. EOS 69:1089 and production of Oscillatoria (Trichodesrnium) spp. In the Glibert PM, Bronk DA (1994) Release of dissolved organic western Sargasso and Caribbean Seas Limnol Oceanogr nitrogen by marine diazotrophic cyanobacteria, Tricho- 22-60-72 desmium spp. Appl Environ Microbiol 6011 1).3996-4000 Carpenter EJ, Romans K (1991) Major role of the cyanobac- Glibert Ph4, Capone DG (1993) Mineralization and assimila- terium Trichodesmium in nutrient cycling in the North tion in aquatic, sediment, and wetland systems. In: Atlantic Ocean. Science 254:1356-1358 Knowles R, Blackburn TH (eds) Nitrogen isotope tech- Carpenter EJ, Bergman B, Dawson R, Siddiqui PJA, Soder- niques. Academic Press, Inc. New York, p 243-271 back E, Capone DG (1992) Glutamine synthetase and Goering JJ, Dugdale RC, Menzel DW (1966) Estimates of in nitrogen cycling in colonies of the marine diazotrophic situ rates of nitrogen uptake by Trjchodesmium sp. in the cyanobacteria Trichodesmium spp Appl Envi.ron Micro- tropical Atlantic Ocean. Limnol Oceanogr 11 614-620 biol 58:3122-3129 Guerrero MG, Lara C (1987) Assin~ilationof inorganic nitro- Carpenter EJ, Capone DG, Fry B, Harvey HR (1997) Biogeo- gen. In: Fay P, Van Baalen C (eds) The cyanobacteria. chemical tracers of the marine cyanobacterium Tri- Elsevier Science Publishers, New York, p 163-186 chodesmium. Deep-Sea Res 44:27-38 Janson S, Carpenter EJ, Bergman B (1994) Compartmentali- Carpenter EJ, Montoya JP, Burns J, Mulholland MR, Subra- sation of nitrogenase in a non-heterocystous cyanobac- maniam A, Capone DG (1999) Extensive bloom of a N2- terium: Trichodesrnium contorturn. FEMS Microbiol Lett fixing diatom/cyanobacterial association in the troplcal 118-9-14 Atlantic Ocean Mar Ecol Prog Ser 185.273-283 Karl DM, Letelier R,Hebel DV, Bird DF, Winn CD (1992) Tri- Checkley DM, Miller CA (1989) Nitrogen isotope fractiona- chodesmium blooms and new nitrogen in the North Pacific tion by oceanic zooplankton. Deep-Sea Res 36:1449-1456 Gyre. In: Carpenter EJ, Capone DG. Rueter JG (eds) Chen YB, Zehr JP, Mellon M (1996)Growth and nitrogen fixa- Marine pelagic cyanobacteria:Trjchodesmiurn and other tion of the diazotrophic filamentous nonheterocystous diazotrophs. Kluwer Academic Publishers, Dordrecht, cyanobacterium Trichodesmjum sp. IMSlOl in defined me- p 219-237 dia: evidence for a circadian rhythm. J Phycol 32:916-923 Kirchman DL, Keil RG, Wheeler PA (1989)The effect of amino Cowie GL, Hedges JI (1992) Improved amino acid quantifica- acids on ammonium utihzation and regeneration by het- tion in environmental samples: charge-matched recovery erotroph~cbacteria In the subarctic Pacific. Deep-Sea Res standards and reduced analysis time. Mar Chem 37: 36:1763-1776 223-238 Kramer JG. Wyman M, Zehr JP. Capone DG (1996) Die1 vari- Devassy VP (1987) Trichodesmiurn red tides in the Arabian ability in transcription of the structural gene for glutarnine Sea. In: Qasim SZ (ed) Contributions in marine sciences, synthetase (glnA) in natural populations of the marine dia- Sastyabdapurti felicitation Volun~e p 61-66 zotrophlc cyanobacterlum Trichodesmium thiebautij. Eppley R\Y, Petersen BJ (1979) Part~culateorganic matter flux FEMS Microbiol Ecol 21.187-196 and plankton~cnew production in the deep ocean. Nature Lee KIT,Joseph CM, Meeks JC (1988) Glutanline synthetase 282:677-680 specific activity and protein concentration in symbiotic Flores E, Herrero A (1994) Assimilatory nitrogen metabolism Anabaena associated with Azolla caroliniana. Antonie and its regulation. In: Bryant DA (ed) The molecular Leeuwenhoek 54:345-355 biology of cyanobacteria. Kluwer Acadttmic Publishers, Lowry OH. Passonneau JV (1972) A flexible system of enzy- Dordrecht, p 487-517 matic analysis. Academic Press, Inc., New York Flynn KJ (1988) Some practical aspects of measurements of Lowry OH, Carter J, Ward JB, Glaser L (1971) The effect of car- dissolved free amino acids in natural waters and within bon and nitrogen sources on the level of metabolic inter- microalgae by the use of HPLC. Chem Ecol3:369-293 mediates in Escl~erichiacoli. J Biol Chem 246:6511-6521. Mulholland & Capone: Nitrogen metabolism in Trichodesmium spp. 49 Mague TH, Mague FC, Holm-Hansen 0 (1977) Physiology Pedersen H, Lomstein BA, Blackburn TH (1993) Evidence for and chemical composition of nitrogen-fixing phytoplank- bacterial urea production in marine sediments. FEMS ton in the central North Pacific Ocean. Mar Biol 41: Microbiol Ecol 12.51-59 213-223 Prufert-Bebout L, Paerl HW, Lassen C (1993) Growth, nitro- Marumo R (1975) An outline of studies in the community of gen fixation, and spectral attenuation in cultivated Tri- marine pelag~cblue-green algae. In: Marumo R (ed) Stud- chodesmium species. Appl Environ Microbiol 59(5): ies on the community of marine pelagic blue-green algae, 1367-1375 1972-1974. Ocean Research Institute, Tokyo, p 1-16 Saino T (1977) Biological nitrogen fixation in the ocean with McCarthy JJ, Carpenter EJ (1979) OsciLIatoria (Tricho- emphasis on the nitrogen fixing blue-green alga Tricho- desmium) thiebautii (Cyanophyta) in the central North desmium and its significance in the nitrogen cycling in the Atlantic Ocean. J Phycol 15:75-82 low latitude sea areas. PhD thesis, Ocean Research Insti- Mulholland MR, Glibert PM, Berg GM, Van Heukelem L, tute, University of Tokyo Pantoja S, Lee C (1998) Extracellular amino acid oxidation Saino T, Hattori A (1978) Die1 variation in nitrogen fixation by mcroplankton: a cross-ecosystem comparison. Aquat by a marine blue-green alga, Tiichodesmium thiebautii. Microb Ecol 15141-152 Deep-Sea Res 251259-1263 Mulholland MR. Ohki K, Capone DG (1999) N utilization and Senior PJ (1975) Regulation of nitrogen metabolism in metabolism relative to patterns of N, fixation in cultures of Escherichja coli and Klebsiella aerogenes: studies with the Trichodesmium NIBB1067. J Phycol (in press) continuous-culture technique. J Bacteriol 123:407-418 Nausch M (1996) Microbial activities on Trichodesmium col- Slawyk G, Rodier M (1988) Biosynthetically active glutamine onies. Mar Ecol Prog Ser 141:173-181 synthetase in the marine diatom Phaeodactylum tricornu- Ohki K (1999) A possible role of temperate virus(es) in the reg- tom: optimization of the forward-reaction assay. Mar Biol ulation of Trichodesmium biomass In: Charpy L, Larkum 97:269-274 AWD (eds) Marine cyanobacteria. Bulletin de 1'Institut Slawyk G, Ralmbault P, L'Helguen S (1990) Recovery of urea Ockanographique, Monaco. Special Issue No. 19 (in press) nitrogen from seawater for measurement of 15N abun- O'Neil JM, Metzler PM, Glibert PM (1996) Ingestion of "N,- dance in urea regeneration studies using the isotope- labelled Trichodesmium spp. and ammonium regenera- dilution approach. Mar Chem 30:343-362 tion by the harpacticoid copepod Macrosetella gracilis. Stadtman ER, Smyrniotis PZ, Davis TN, Wittenberger ME Mar Rio1 125:89-96 (1979) Enzymatic procedures for determining the average Paerl HW, Bebout BM, Prutert LE (1989) Bacterial associations state of adenylylation of Eschenchia col1 glutamine syn- with marine Oscillatoria sp. (Trich~des~iumsp.) popu- thetase. Anal Biochem 951275-285 lation~.ecophysiological implications. J Phycol25:773-784 Thoresen SS, Dortch Q, Ahmed S1 (1982) Comparison of Palenik BP, Morel FMM (1990a) Amino acid utilization by methods for extracting intracellular pools of inorganic marine phytoplankton: a novel mechanism. Limnol Oce- nitrogen from marine phytoplankton. J Plankton Res 4: anogr 35:260-269 695-704 Palenik BP, Morel FMM (1990b) Comparison of cell-surface Turner NE, Robinson SJ, Haselkorn R (1983) Different pro- L-amino acid oxidases from several marine phytoplank- moters for the Anabaena glutamine synthetase gene dur- ton. Mar Ecol Prog Ser 59:195-201 ing growth using molecular or fixed nitrogen. Nature Palenik BP, Kieber DJ. Morel FMM (1988) Dissolved organic 306:337-342 nitrogen use by phytoplankton: the role of cell-surface Wyman M, Zehr JP, Capone DG (1996) Temporal variability enzymes. Biol Oceanogr 6:347-354 in nitrogenase gene expression in a natural population of Pantoja S, Lee C (1994) Cell-surface oxidation of amino acids the marine cyanobacterium Trichodesmium thiebautii. in seawater. Limnol Oceanogr 39:1718-1726 Appl Environ Microbiol 62:1073-1075 Pantoja S, Lee C, Marecek JF, Palenik BP (1993) Synthesis Zehr JP, Wyman M. Miller V, Duguay L, Capone DG (1993) and use of fluorescent molecular probes for measuring Modification of the Fe protein of nitrogenase in natural cell-surface enzymatic oxidation of amino acids and amines populations of Tricl~odesmium thiebautji. Appl Environ in seawater. Anal Biochem 211:210-218 Microbiol 59:669-676 Editorial responsibility: Kenneth Tenore (Contributing Submitted: November 3, 1998; Accepted: April 27, 1999 Editor), Solomons, Maryland, USA Proofs received from author(s): October 26, 1999