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Applied and Environmental Microbiology, February 2004, p. 765-770, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.765-770.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

N₂ Fixation by Unicellular Bacterioplankton from the Atlantic and Pacific Oceans: Phylogeny and In Situ Rates

Luisa I. Falcón,^1* Edward J. Carpenter,² Frank Cipriano,³ Birgitta Bergman,⁴ and Douglas G. Capone⁵

Marine Sciences Research Center, Stony Brook University, Stony Brook, New York 11794-5000,¹ Romberg Tiburon Center for Environmental Studies, San Francisco State University, Tiburon, California 94920,² Conservation Genetics Laboratory, Biology Department, San Francisco State University, San Francisco, California 94132,³ Botanical Institute, Stockholm University, S-106-91 Stockholm, Sweden,⁴ Wrigley Institute for Environmental Studies, University of Southern California, Los Angeles, California 90089⁵

Received 11 July 2003/ Accepted 4 November 2003

Top Abstract Introduction Materials and Methods Results and Discussion References

 
N₂-fixing proteobacteria ( and ) and unicellular cyanobacteria are common in both the tropical North Atlantic and Pacific oceans. In near-surface waters proteobacterial nifH transcripts were present during both night and day while unicellular cyanobacterial nifH transcripts were present during the nighttime only, suggesting separation of N₂ fixation and photosynthesis by unicellular cyanobacteria. Phylogenetic relationships among unicellular cyanobacteria from both oceans were determined after sequencing of a conserved region of 16S ribosomal DNA (rDNA) of cyanobacteria, and results showed that they clustered together, regardless of the ocean of origin. However, sequencing of nifH transcripts of unicellular cyanobacteria from both oceans showed that they clustered separately. This suggests that unicellular cyanobacteria from the tropical North Atlantic and subtropical North Pacific share a common ancestry (16S rDNA) and that potential unicellular N₂ fixers have diverged (nifH). N₂ fixation rates for unicellular bacterioplankton (including small cyanobacteria) from both oceans were determined in situ according to the acetylene reduction and ¹⁵N₂ protocols. The results showed that rates of fixation by bacterioplankton can be almost as high as those of fixation by the colonial N₂-fixing marine cyanobacteria Trichodesmium spp. in the tropical North Atlantic but that rates are much lower in the subtropical North Pacific.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Growth rates of plankton in open ocean surface waters are often limited by the availability of reduced forms of N. New combined N enters surface waters either by advection, diffusion of NO₃ from deep water, or biological N₂ fixation (15). The last pathway can be significant in tropical and subtropical seas, where large cyanobacteria, Trichodesmium spp., have been considered the major organisms responsible (8, 9, 11). Ongoing research to identify other sources of N₂ fixation and alternate pathways of reduced N to the trophic chain in the vast oceanic basins has pointed out the potential role of certain N₂-fixing unicellular bacterioplankton, which could have a significant impact on global biogeochemistry of N and C (14, 16, 44).

Previous research has identified the presence of fragments of the unicellular cyanobacterial and proteobacterial nifH genes (which encode one of the peptide molecules that form the dinitrogenase reductase subunit of nitrogenase, the enzyme responsible for N₂ fixation [31]) in the subtropical North Pacific and tropical North Atlantic oceans (16, 43, 44). Preliminary data on N₂ fixation rates for the 10- to 0.2-µm-size fraction of the bacterioplankton have suggested that they could make an important contribution to the global N cycle (14, 44). These studies used ¹⁵N₂ to measure 24-h N₂ fixation rates in 10-µm-pore-size-prefiltered water from the subtropical North Pacific. Also, N₂ fixation rates were estimated based on unicellular phytoplankton cell numbers that came from literature values (6). Cyanobacteria are autotrophs and generate ATP necessary for N₂ fixation through photosynthesis; unicellular cyanobacteria that fix N₂ need to do so during the night (2) since nitrogenase, the enzyme that catalyzes N₂ fixation, is inhibited by O₂ (15). Thus, it is important to obtain nitrogenase activity rates for the daytime and the nighttime.

In order to better define the in situ rates of N₂ fixation for the unicellular bacterioplankton community in the tropical North Atlantic and subtropical North Pacific oceans, we sampled at different depths throughout diel cycles for different seasons by the acetylene reduction and ¹⁵N₂ protocols with slight variations (7, 10). Further, we coupled the N₂ fixation activity to the phylogenetic characterization of the populations responsible throughout the analysis of nifH transcripts. Here we demonstrate that potential N₂-fixing unicellular bacterioplankton differ between the tropical North Atlantic and North Pacific; we also show that nifH transcripts of unicellular cyanobacteria occur during the nighttime only while those for proteobacteria are present during both the day and the night. Major differences are also found in N₂ fixation rates between the two oceans; in the tropical North Atlantic, the unicellular bacterioplankton activity is almost as high as that of the major oceanic N₂ fixers Trichodesmium spp. but in the tropical North Pacific is much lower. Our findings provide unique and new information essential to understanding N and C cycles in the Atlantic and Pacific oceans.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Seawater sampling.
Water samples were collected during two cruises to the tropical North Atlantic (summer 2001 and spring 2002) and during one cruise to the tropical North Pacific (fall 2002). Our research area in the Atlantic was concentrated between 7°10'23"N to 12°13'78"N and 45°29'60"W to 55°55'38"W, and in the Pacific it was in proximity to station ALOHA, 22°75'N to 158°00'W (19). Samples were obtained from discrete depths following a light level range of 100, 50, 25, and 1% which was determined previous to each conductivity-temperature-depth (CTD)-rosette system and which corresponded approximately to surface, 25, 50, and 100 m; also, samples were taken at the depth of increase in the nitracline, which was usually around 150 m. Seawater was gravity filtered from Niskin 10-liter bottles through 20-µm-pore-size mesh (tropical North Pacific) and 10-µm-pore-size polycarbonate (PCTE) membranes (Millipore Corporation, San Jose, Calif.) (tropical North Atlantic) to eliminate larger diazotrophs (e.g., Trichodesmium spp.) and collected in Nalgene bottles. In the Pacific, we observed by epifluorescence microscopy that large unicellular cyanobacteria (7 µm) clustered together and clogged the 10-µm-pore-size membranes.

Nitrogenase activity.
The filtrate from each light depth (4 liters) was concentrated onto 0.2-µm-pore-size membranes (47-mm diameter; polycarbonate PCTE; Millipore). Cell concentrates were diluted by washing the filters with 0.2-µm-pore-size-filtered seawater, to the volume necessary to assay nitrogenase activity by the ¹⁵N₂ and acetylene reduction protocols (7, 10). Each 0.2-µm-pore-size membrane was screened under the epifluorescence microscope to verify that the cells had been washed off. Final enrichments were about 130- to 160-fold. ¹⁵N₂ experiments were carried out during the summer 2001 cruise with 14 ml of cell concentrate (no headspace left in 14-ml vials) during 24 h and incubated with 9 µl of ¹⁵N₂ (data from three stations, n = 3 per depth per station). Experiments were ended by filtering the contents of each vial onto a combusted glass fiber filter that was folded and left to dry in an oven at 60°C before being analyzed with a mass spectrometer. During the spring 2002 (Atlantic) and fall 2002 (Pacific) cruises, the diel and depth variations of N₂ fixation were examined by the acetylene reduction procedure (n = 18 per depth [Atlantic]; n = 24 per depth [Pacific]). Experiments were started with injection of acetylene into serum vials (14 ml) which contained 10 ml of cell concentrate; time zero values were registered immediately after. Samples were run for 24 h, and ethylene readings were carried out approximately every 4 to 6 h, preceded by ethylene standard quantifications. Twenty-four-hour N₂ fixation rates were integrated over the water column depth for the times when nitrogenase activity was observed. Each depth experimental sample (n = 3) was left inside a bag that mimicked its corresponding light level in a flowing seawater on-deck incubator. Control assays were carried out in parallel with 0.2-µm-pore-size-filtered seawater.

Cloning and sequencing.
After completion of the 24-h nitrogenase activity experiments, daytime and nighttime samples were filtered onto 0.2-µm-pore-size Durapore membrane filters (25-mm diameter; Millipore) for RNA extraction (41) with an RNeasy kit; residual amounts of DNA were removed using an RNase-Free DNase set (Qiagen, Valencia, Calif.). An Access reverse transcriptase PCR kit (Promega, Madison, Wis.) was used to amplify cDNA for nifH genes (357-bp fragment) (42). The reverse transcription reaction was carried out for 30 min using 1 µM primer nifH 3, 28 µl of H₂O, 10 µl of 5x avian myeloblastosis virus buffer, 1 µl deoxynucleoside triphosphates (10 mM each), 1 µl of avian myeloblastosis virus reverse transcriptase, and 1 µl of DNA-free RNA (41). Four liters of prefiltered water was collected onto 0.2-µm-pore-size Durapore membrane filters (25-mm diameter; Millipore), and DNA was extracted according to standard protocols (26, 33). Primers (CYA 359F and 781b) that amplify a conserved 16S ribosomal DNA (rDNA) cyanobacterial region (approximately 422-bp fragment) (29) were used in a typical PCR. Purified nifH and 16S rDNA products were ligated with the pGEM-T Easy vector system (Promega) (33). Reaction mixtures (50-µl final volume) contained the following: 33.5 µl of RNA-DNA-free water, 5 µl of PCR buffer, 5 µl (16S rDNA) or 8 µl (nifH) of MgCl₂ (25 mM), 1 µl of deoxynucleoside triphosphate mix, 2 µl of each primer (12.5 µM) (29, 42), 1 µl of template (cDNA for nifH), and 0.5 µl of Taq DNA polymerase (Promega). The thermal cycle for amplification of 16S rDNA was as follows: 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min for 35 cycles. The thermal cycle for amplification of nifH was as follows: 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min for 30 cycles. Purified amplified clone products were cycle sequenced with an ABI Prism BigDye kit (Perkin-Elmer, Foster City, Calif.).

Phylogenetic reconstruction analysis using PAUP*4.0b8 (Sinauer Associates Inc., Sunderland, Mass.) included parsimony bootstrapping (250 replicates, random addition initial trees). Distance- and maximum-likelihood-based analysis (data not shown) gave topologies nearly identical to that of the parsimony analysis. The resulting 72-taxon nifH data set included 217 parsimony-informative characters. The resulting 32-taxon 16S rDNA summarized data set (1) included 110 parsimony-informative characters.

Cell counts.
Unicellular cyanobacteria cell counts were done on board within an hour of collection, using an epifluorescence compound microscope (Zeiss Axioscope) at 1,000x. Seawater filtrates were collected onto 0.2-µm-pore-size PCTE membranes (47-mm diameter; Millipore) and placed in microscope slides for observation. Blue and green excitations were used to observe phycoerythrin and chlorophyll a epifluorescence signals, respectively (25).

Nucleotide sequence accession number.
The sequences generated in this study have been deposited in the GenBank database under the indicated accession numbers: tropical North Atlantic nifH, AF536983 to AF536986 and AY191972 to AY191976; tropical North Atlantic 16S rDNA, AY191920, AY191921, and AY191923 to AY191942; subtropical North Pacific nifH, AY191943 to AY191966, AY191969, and AY191971; subtropical North Pacific 16S rDNA, AY191919 and AY191904 to AY191922.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Phylogenetic analysis.
Partial nifH sequences of both unicellular cyanobacteria and proteobacteria clustered separately depending on the ocean of origin (Fig. 1). Most of the cDNA nifH sequences obtained for the North Atlantic in April 2002 clustered mainly (32 clones out of a 37-clone library) with unicellular cyanobacterial sequences for this same region in August 2001 published previously (16) as well as with the unicellular cyanobacterial diazotrophs Synechocystis sp. strain WH8501, which was isolated in 1988 from the tropical North Atlantic (39), and Cyanothece strain ATCC 51142, isolated from the Gulf of Mexico (32). The remaining five Atlantic cDNA nifH clones clustered with -proteobacterium-like sequences. Five unique sequences from the subtropical North Pacific (10 clones out of a 34-clone library) clustered with unicellular cyanobacteria, which had been seen only in the North Pacific in May (44). Most of the Pacific nifH cDNA clone sequences (including all of those obtained from 100-m samples from incubations from 1200 to 1800 h for activity) clustered with -proteobacteria, and one unique sequence clustered with -proteobacteria. We cannot assign the observed nitrogenase activity with certainty to one group of bacteria; nevertheless, we obtained only -proteobacterium-type nifH transcripts from daytime incubations in the subtropical North Pacific; - and -proteobacteria and unicellular cyanobacterium-like nifH transcripts were obtained during the nighttime incubations (both oceans). The 16S rDNA cyanobacterial sequences from both oceans clustered together and with other proposed N₂-fixing unicellular cyanobacteria (Fig. 2) (Synechocystis sp. strain WH8501). This field observation is consistent with previous studies that have proposed that N₂-fixing unicellular cyanobacteria obtain ATP necessary for N₂ fixation during the day through C fixation and temporally separate O₂ production from nitrogenase activity (17, 23, 27, 28, 32, 35, 36, 37). Also, since proteobacteria are heterotrophic, their N₂ fixation activity is not controlled by the day-night cycles of C fixation. Microanaerobic environments can occur in the surroundings of cells that are respiring, which could provide the environments adequate to carry out N₂ fixation during the day (19, 30). In any event, the in situ N₂ fixation rates measured were greater during the nighttime, and in fact, only the subtropical North Pacific showed a small daytime N₂ fixation activity. Nevertheless, even though the obtained cyanobacterial nifH transcripts and 16S rDNA sequences clustered together with those from known N₂-fixing unicellular cyanobacteria (Synechocystis sp. strain WH8501 and Cyanothece strain ATCC 51142), we cannot assume that unicellular cyanobacteria analyzed in this study were N₂ fixers. In fact, since the 16S rDNA region that we sequenced is only approximately 422 bp in length, it is not yet clear how closely related populations from the Atlantic and Pacific really are.

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FIG. 1. Phylogenetic reconstruction (maximum parsimony) for nifH gene sequences from bacterioplankton in the tropical North Atlantic and subtropical North Pacific oceans. Asterisks indicate sequences obtained as part of this study. Daytime and nighttime transcripts are indicated by open and solid circles, respectively.

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FIG. 2. Summary of phylogenetic reconstruction (maximum parsimony) for 16S rDNA sequences from unicellular cyanobacteria in the tropical North Atlantic and subtropical North Pacific oceans. Asterisks indicate sequences obtained as part of this study.

The differential clustering of unicellular cyanobacteria from the tropical North Atlantic and subtropical North Pacific based on 16S rDNA and nifH phylogenies suggests a common ancestry (16S rDNA) and long-term genetic divergence (nifH) between populations of diazotrophic unicellular cyanobacteria in the tropical North Atlantic and North Pacific. Further, it is interesting to relate unicellular cyanobacterial nifH sequences in the same region of the tropical North Atlantic in 1998 (43), in the summer of 2001 (16), and in the spring of 2002, which could suggest that these unicellular cyanobacteria may be a permanent component of the spring-summer stratified water column in this area of the tropical oceans.

In situ rates of N₂ fixation.
We measured unicellular bacterioplankton N₂ fixation in the mixed surface water layer of the tropical North Atlantic in the summer of 2001 (when we observed a strong thermocline and nutricline [http://biology.usc.edu/bc/]) and during spring 2002 (which again showed a stable surface water mixed layer [http://biology.usc.edu/bc/]). Nitrogenase activity was observed only during the night both in the summer of 2001 and in the spring of 2002 (Fig. 3). Integrated water column rates for 10 h of nighttime nitrogenase activity in the tropical North Atlantic averaged 47 µmol of N m^-2 day^-1 in summer 2001 and 37 µmol of N m^-2 day^-1 in spring 2002. Rates of N₂ fixation by Trichodesmium spp. were measured in parallel with our experiments in the tropical North Atlantic during the summer 2001 cruise (D. G. Capone, personal communication). For that cruise, the average rate for 30 stations was 62 ± 21 (standard error) µmol of N m^-2 day^-1. A cruise in the same vicinity in February 2001 found average rates of 167 ± 49 (standard error) µmol of N m^-2 day^-1 (n = 23) (D. G. Capone, personal communication). Thus, unicellular bacterioplankton N₂ fixation activity at several stations in the tropical North Atlantic was about equivalent to that of Trichodesmium spp. in the summer of 2001 and about 20% of Trichodesmium spp. N₂ fixation activity in the spring of 2002.

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FIG. 3. Distribution of average rates of in situ N2 fixation (picomoles of N liter-1 hour-1) by bacterioplankton by depth for the tropical North Atlantic (solid symbols) and subtropical North Pacific (open symbols). 15N2 24-h incubations in summer 2001 are shown by . Symbols indicate periods of incubations and reading time in gas chromatography: , 1800 to 2200 h; , 2200 to 0200 h; and •, 0200 to 0600 h for spring 2002, tropical North Atlantic; , 1200 to 1800 h; , 1800 to 2400 h; and , 2400 to 0600 h for fall 2002, subtropical North Pacific. Error bars represent standard errors. Only readings where nitrogenase activity was detected are shown.

During the fall of 2002, in the subtropical North Pacific, we observed N₂ fixation by unicellular bacterioplankton primarily during the night; measurable daytime nitrogenase activity was also observed (Fig. 3). Integrated water column N₂ fixation rates for 12 h of nitrogenase activity averaged 2.2 µmol of N m^-2 day^-1. The N₂ fixation rates for the fall in the subtropical North Pacific are only 2% of those previously estimated in the summer of 2000 based on cell abundances of the 3- to 20-µm phytoplankton size fraction (44) and approximately 11% of those estimated from 24-h incubations with ¹⁵N₂-enriched water in November 2000 (14). In the subtropical North Pacific, reported rates of N₂ fixation by Trichodesmium spp. (8) are 84 ± 50 µmol of N m^-2 day^-1; thus, unicellular bacterioplankton would increase these rates by only 2.6%.

Unicellular cyanobacterial cell abundances.
Unicellular cyanobacterial abundances were an order of magnitude higher in the Atlantic than in the Pacific, peaking from surface to 25 m in both oceans (Fig. 4); cells were larger in the Pacific (3 to 7 µm) than in the Atlantic (2.5 µm). The cell abundances reported here included all unicellular cyanobacteria (2.5 µm, Atlantic; 3 to 7 µm, Pacific), both the potential N₂ fixers and the non-N₂ fixers. The fluorochrome system used (24) does not work on unicellular cyanobacteria since these have a similar fluorescent signal with or without a positive in situ immunolocalization of cells expressing nitrogenase.

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FIG. 4. Distribution of average cell counts of unicellular cyanobacteria by depth, in the tropical North Atlantic (solid symbols; 2.5 µm) and subtropical North Pacific (open symbols; 3 and 7 µm) oceans. Error bars represent standard errors.

Previous studies have shown that total bacterial cell numbers in marine oligotrophic environments tend to be balanced over time by bacterivores and viruses and are comparable between the tropical North Atlantic and the subtropical North Pacific (12).

Our data suggest a trend of increased unicellular cyanobacterial abundance and N₂ fixation rates in the tropical North Atlantic compared to those in the subtropical North Pacific (Fig. 3 and 4); this is an interesting observation, since it has been suggested that unicellular diazotrophic cyanobacteria might be major diazotrophs in oligotrophic marine ecosystems (44).

We hypothesize that differences in N₂ fixation rates of unicellular bacterioplankton and cell abundances of coccoid cyanobacteria (>2.0 µm) observed between oceanic basins are in part due to the larger eolian Fe flux to the North Atlantic (0.2 to 0.8 µmol of Fe m^-2 day^-1) (40) and to the high Fe/P ratio present in this area, which have been suggested (4, 5, 38) to favor N₂ fixation in comparison to the North Pacific Ocean (20) (0.08 to 0.16 µmol of Fe m^-2 day^-1) (40). Nevertheless, to date, Fe cell quotas for proteobacterial and unicellular cyanobacterial diazotrophs are needed, as are field measurements of the Fe/C ratio of proteobacterial and unicellular cyanobacterial diazotrophs (22). The tropical North Pacific (20, 21) has low light levels and deeper mixed layers that show a pattern of diminished N₂ fixation rates (14). In the summer, the thermocline reaches its maximum in tropical oceans, bringing stability and increasing surface water temperatures, which could have a direct effect on the diazotrophic activity of unicellular bacterioplankton.

The magnitude of N₂ fixation by small unicellular bacterioplankton, the stability of their populations over time, and the differences found between the tropical North Atlantic and the subtropical North Pacific (3, 5, 8, 13, 18, 20, 21, 34, 38, 40) suggest that the marine N cycle of these spatially extensive ecosystems has more components, and is more complex, than previously thought.

 
This work was funded by the National Science Foundation. L.I.F. is funded through CONACyT (Mexico) and NSF grants to E.J.C. Financial support from the Swedish Research Council and STINT (Sweden) is acknowledged (B.B.).

 
* Corresponding author. Present address: Romberg Tiburon Center for Environmental Studies, San Francisco State University, 3152 Paradise Dr., Tiburon, CA 94920. Phone: (415) 338-3737. Fax: (415) 435-7121. E-mail: lfalcon{at}sfsu.edu.

Top Abstract Introduction Materials and Methods Results and Discussion References

 

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Applied and Environmental Microbiology, February 2004, p. 765-770, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.765-770.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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