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Applied and Environmental Microbiology, September 1998, p. 3444-3450, Vol. 64, No. 9
Department of Biology, Rensselaer Polytechnic
Institute, Troy, New York 12180-3590
Received 27 April 1998/Accepted 24 June 1998
Oligotrophic oceanic waters of the central ocean gyres typically
have extremely low dissolved fixed inorganic nitrogen concentrations, but few nitrogen-fixing microorganisms from the oceanic environment have been cultivated. Nitrogenase gene (nifH)
sequences amplified directly from oceanic waters showed that the open
ocean contains more diverse diazotrophic microbial populations and more
diverse habitats for nitrogen fixers than previously observed by
classical microbiological techniques. Nitrogenase genes derived from
unicellular and filamentous cyanobacteria, as well as from the The productivity of the oceans
controls the fluxes of many biogeochemically important compounds,
including the rate of exchange of carbon dioxide between the open ocean
and the atmosphere. In turn, oceanic carbon fixation is limited by the
bioavailability of nutrients, including nitrogen, phosphorus, and iron
(9, 10, 20). In contrast to the biogeochemical cycles of
phosphorus and iron, nitrogen is present in relatively high
concentrations in seawater as gaseous N2. Gaseous nitrogen
is available only to microorganisms with the capability of biological
nitrogen fixation, the reduction of atmospheric N2 to
ammonium. Although large areas of the world's oceans are virtually
devoid of fixed dissolved inorganic nitrogen and primary production may
be nitrogen limited, very few species of nitrogen-fixing
organisms have been identified or isolated from the plankton.
Trichodesmium, a filamentous aggregate-forming cyanobacterium, is an abundant diazotroph in tropical and subtropical waters (3, 5), but few other examples of diazotrophs
from the open ocean are known (21, 35). The seeming low
diversity of known nitrogen-fixing organisms in the open ocean stands
in stark contrast to the presumptive nitrogen limitation in the
world's oceans and presents an evolutionary paradox.
Recently, biological nitrogen fixation has gained recognition as an
important source of nitrogen for supporting oceanic primary production
(3, 11, 18, 22). The nitrogen budget for the Atlantic Ocean
does not balance because a source of nitrogen cannot be accounted for
by current knowledge of fluxes and pools of nitrogen, even after
including nitrogen fixation by Trichodesmium
(22). It is speculated that rates of nitrogen fixation by
known diazotrophic organisms have been underestimated (17),
or as yet unidentified diazotrophic organisms are active in the ocean
(18). Conventional nitrogenase, the enzyme that catalyzes
biological dinitrogen reduction to ammonium, is composed of two highly
conserved proteins: the iron (Fe) protein (encoded by the
nifH gene) and the molybdenum iron (MoFe) protein (encoded
by the nifDK genes). The nitrogenase enzyme is present in
diverse lineages of prokaryotes and is generally believed to be ancient
(38). Evolutionarily conserved amino acid sequences within
the nifH (which encodes the Fe protein component of
nitrogenase) gene have been exploited to design PCR primers to detect
the genetic potential for nitrogen fixation in the marine environment
(39). With this approach, the diversity of nitrogen-fixing microorganisms in oceanic water and marine plankton was determined. This report shows that there are far more diverse nitrogen-fixing populations and diverse habitats which can support nitrogen fixation in
the open ocean than previously documented.
Sample types, sampling locations, depths, and sampling dates are
shown in Table 1. Samples were collected
from the Bermuda Atlantic Time Series (BATS) and Hawaii Ocean Time
Series (HOT) stations during routine sampling. Samples were also
collected during cruises on ships of opportunity in the Atlantic and
Pacific oceans and Caribbean Sea. Water was collected at depths ranging from 0 to 200 m, using Niskin water sampling bottles. Samples for
picoplankton (0.22- to 20-µm size fraction) DNA extraction were
prefiltered (through 20-µm nylon Nytex mesh) to remove
Trichodesmium and zooplankton, and the microbial assemblages
were filtered onto Gelman Supor 0.22-µm-pore-size filters. Water
samples were typically 2 to 4 liters in volume except for the Atlantic
equatorial samples and samples collected near the Bahama Islands, where
20 liters of water was concentrated in an Amicon DC10L tangential flow
system fitted with a hollow-fiber (30,000-molecular-weight) cartridge. The concentrate from the tangential flow system (approximately 250 ml)
was filtered onto Gelman filters as described above.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
New Nitrogen-Fixing Microorganisms Detected in
Oligotrophic Oceans by Amplification of Nitrogenase
(nifH) Genes
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
and
subdivisions of the class Proteobacteria, were found in
both the Atlantic and Pacific oceans. nifH sequences that
cluster phylogenetically with sequences from sulfate reducers or
clostridia were found associated with planktonic crustaceans.
Nitrogenase sequence types obtained from invertebrates represented
phylotypes distinct from the phylotypes detected in the picoplankton
size fraction. The results indicate that there are in the oceanic
environment several distinct potentially nitrogen-fixing microbial
assemblages that include representatives of diverse phylotypes.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
TABLE 1.
Classification of major types of nifH
sequences obtained from marine picoplankton and zooplankton
samples, showing sample source and location
Zooplankton were collected by 150-µm net tow at a depth of 2 m in the Gulf of Mexico near Florida (29°N, 84°W) in the fall of 1994. Live adult individuals of the calanoid copepods Labidocera aestiva and Acartia tonsa were sorted from the mixed assemblage by using wide-bore pipettes. Sorted individuals were washed in prefiltered (through a 0.2-µm-pore-size filter) seawater prior to preservation.
All samples were frozen in 10 mM Tris (pH 8.0)-100 mM EDTA (pH 8.0) until analyzed. DNA was extracted from the filter and zooplankton samples by using a slight modification of the method of Giovannoni et al. (13).
Nitrogenase Fe protein genes (nifH) were amplified from picoplankton- and zooplankton-derived genomic DNA, using the PCR primers of Zehr and McReynolds (41). The samples were amplified by PCR in a mixture containing 4 mM MgCl2 (Promega, Madison, Wis.), the enzyme manufacturer's buffer (Promega), 200 µM deoxynucleoside triphosphates, 100 pmol of each primer, and 2.5 U of Taq polymerase (Promega) in 50-µl volumes for 35 cycles (1 min at 94°C, 1 min at 54°C, and 1 min at 72°C).
The amplified fragments were cloned into Promega pGEM-T vector (Promega). Clones were screened by restriction digestion to identify those with the correct insert, and DNA from the selected clones was used for DNA sequencing by the Sanger dideoxynucleotide chain termination method (28). DNA sequences were obtained on both strands; the consensus sequence was used to determine the deduced amino acid sequence, using the Genetic Data Environment package (30). Deduced amino acid sequences were aligned manually, and the aligned sequences were used for phylogenetic analysis using PHYLIP 3.5c (12) or TREECON for Windows (34).
Nucleotide sequence accession numbers. The sequences obtained in this study were submitted to GenBank under accession no. AF016592 to AF016618 and AF059621 to AF059649 (which includes two new nifH sequences obtained from cultivated isolates of Chromatium purpuratum).
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
|
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Nitrogenase (nifH) genes were amplified from oligotrophic surface water picoplankton from the tropical Atlantic and Pacific oceans and from the Sargasso and Caribbean seas, using PCR and universal primers for the nitrogenase Fe protein gene (41). nifH or nifH-related gene sequences cluster in four major groups (Fig. 1, I to IV [7]). nifH genes not only were found in the picoplankton size fraction from these diverse water samples but also were amplified from marine crustacean zooplankton. Sequences from the marine invertebrate and oceanic picoplankton samples were found only in clusters I (conventional nifH) and group III (divergent nifH from sulfate reducers and clostridia) (Fig. 2 and 3). The nif sequences obtained from free-living picoplankton and invertebrate samples represented diverse heterotrophic and photoautotrophic lineages (Fig. 2 and 3), but more importantly, the sequence types were distinctly different from the two sample types, highlighting the differences in diversity between the two microbial habitats.
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proteobacteria) and clostridia
(gram-positive bacteria). Group IV sequences are from divergent genes
that may not be derived from active nitrogenases.
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, diatom associated; 
,
picoplankton;
,
zooplankton associated.
|
Picoplankton nif genes included sequences clearly derived
from representatives of the and proteobacteria, 
proteobacteria, and unicellular cyanobacteria clades (Table 1; Fig.
2). The phylogeny of nifH clearly distinguishes between
filamentous nonheterocystous, heterocystous, and unicellular strains
(40). nifH sequences from this study clustered
with sequences from group V heterocystous strains and group I
unicellular strains. Unicellular cyanobacterial nifH
sequences were found in low-latitude oligotrophic waters of the
Atlantic and at the BATS and HOT sites (Table 1). The phylotypes
obtained from the Pacific and the Atlantic water samples were
distinctly different, however. The nifH sequences obtained from the Pacific Ocean (sequence types HT1150 and HT1103) cluster with
the group II genera Myxosarcina and Xenococcus,
whereas the sequence type amplified from the Atlantic Ocean (AO11)
clusters more closely with the nifH sequences from
Gloeothece and Cyanothece (previously called
Synechococcus), which are representatives of group I
cyanobacteria (unicellular, dividing in one plane). However, the
resolution of these groups in the nifH tree may currently be
limited by the availability of representative nifH
sequences. The unicellular cyanobacterial nifH genes from
the HOT site were recovered in two different years and from multiple
depths spanning the mixed layer. nifH genes derived
from filamentous species were also detected in the picoplankton size
fraction. Although filamentous cyanobacteria would have been expected
to be largely removed by the prefiltration procedure, the
presence of cells that presumably dissociated from filaments
indicates that filamentous cyanobacteria (nonheterocystous species from
the HOT station and heterocystous species from the BATS site) may
be present in the net plankton.
Unicellular cyanobacteria and prochlorophytes are numerous in the open ocean (8, 16, 36), but it is likely that most of these species are not able to fix atmospheric nitrogen. Only a single open-ocean unicellular nitrogen-fixing strain of cyanobacteria has been found in low-latitude Atlantic waters (37). The few known nitrogen-fixing unicellular cyanobacterial strains (e.g., of the genera Gloeothece and Cyanothece) are distinctly different in morphology from the typically abundant oceanic unicellular cyanobacterial strains and are quite large (5 µm in diameter) (6). Since an oceanic cyanobacterial nitrogen-fixing isolate has been obtained (36) and the results presented here suggest that this type of organism may be more widely distributed in the Pacific and Atlantic Ocean basins than previously documented, these organisms should receive closer attention.
The presence of numerous and proteobacterial (referred to for
simplicity as and ) nifH genes in the picoplankton is consistent with the documentation of numerous and 16S rRNA phylotypes in oceanic waters (23, 29). Noncyanobacterial
nitrogenase genes found in the bulk water include genes closely
related to nifH genes from the genera Azotobacter
and Vibrio (Fig. 2; Table 1). The nifH
phylotypes were found numerous times in samples (35 sequences obtained
that were >98% identical at the nucleotide level [data not shown])
from the equatorial Atlantic waters. Interestingly, this nifH sequence type was not obtained from the zooplankton
samples. Very closely related Vibrio nifH sequences
(31) and strains (32) have been found in
coastal studies, suggesting that these nitrogen-fixing proteobacteria are major nitrogenase gene-containing phylotypes in the
marine environment. A second cluster of nifH sequences
were relatively closely related to sequences obtained from the purple
sulfur bacterium C. purpuratum (Fig. 2). Sequences in this
cluster, as well as the Atlantic Ocean picoplankton samples, were
obtained from the HOT station. Relatively closely related to this
cluster are a group of sequences that group between the
Alcaligenes faecalis nifH sequence and nifH.
These nifH sequences, which may be derived from phylotypes
of proteobacteria, were obtained in the Atlantic Ocean picoplankton
samples as well as from diatom mats collected in the Pacific Ocean
(Fig. 2).
16S genes have been previously discovered in the open ocean and
include the important SAR (Sargasso Sea) clusters (14). The
nifH sequences recovered in this study (Fig. 2) could
even be derived from the SAR-11 phylotypes. Interestingly, the nifH phylotypes have not yet been recovered from the HOT
station, although two sequences (PO3133 and PO3120) were obtained from planktonic Pacific Ocean diatom mats. The Atlantic Ocean nifH phylotype clustered with the Pacific diatom-associated
nifH, but a distinct cluster of nifH
sequences was obtained from BATS (Fig. 2). Although the most closely
related nifH sequences from cultivated organisms are those
from Rhizobium spp., the branches are long relative to the
diversity of nifH sequences on the tree, and thus it is
likely that the nitrogen-fixing proteobacteria represented by these
phylotypes are only distantly related to known organisms.
The nifH sequences obtained from amplification of DNA extracted from planktonic crustacean zooplankton (calanoid copepods L. aestiva and A. tonsa) clustered with different nifH clusters than did sequences obtained from the picoplankton. In comparison to the picoplankton samples, the zooplankton nifH gene sequences represented distinct lineages, with abundant sequences clustering with sequences from sulfate reducers and clostridia (group III nitrogenase [Fig. 3]). Interestingly, sequences in this cluster obtained from direct DNA extracts of marine zooplankton were also obtained from microbial enrichments initiated with zooplankton biomass (2). The diverse set of nifH genes amplified from individual copepods (A. tonsa and L. aestiva) collected from Gulf of Mexico waters (Fig. 1 and 2; Table 1) suggests that nitrogen-fixing microorganisms may be associated with the exoskeleton and/or the gut tract of planktonic crustacea. With one exception, all of the nifH genes obtained by amplification from zooplankton were distinctly different from the nifH sequences obtained from the picoplankton. Although the two zooplankton sequences, GM22 and GM26, form a distinct cluster, there is some similarity of sequences GM22 and GM26 obtained from the calanoid copepod A. tonsa and the sequences AO14 and BH1132 obtained from the Atlantic Ocean and Bahama Island picoplankton samples.
The nifH sequences obtained from zooplankton were
distantly related to the nifH sequences derived from the
picoplankton (Fig. 2). It is noteworthy that genes in one of the
clusters of nifH genes recovered from the zooplankton
are unique with respect to nifH genes described thus far.
The nifH sequences in this cluster contain a 12-amino-acid
residue insertion (Fig. 4). It is
unlikely that these nifH genes are pseudogenes. The reading
frame is maintained throughout the insertion, and the physical location
of the insertion in the three-dimensional structure of the Fe protein
is on the surface of the protein (data not shown). This loop would not
likely interfere with nucleotide binding or the FeS cluster, and it is not near the interface where the MoFe protein docks with the Fe protein. The chance that such an extensive insertion would have occurred as an evolutionary event in a pseudogene without associated extensive accumulation of mutations in the rest of the amino acid sequence is extremely low. Phylogenetically, the unique sequences are
relatively closely related to other proteobacterial sequences (Fig. 2).
Thus, the evidence suggests that the nifH sequences were
derived from potentially active, novel nitrogenases.
|
Most of the other nitrogenase gene sequences obtained from zooplankton clustered with a diverse clade that includes sequences from sulfate-reducing bacteria and from clostridia (Fig. 3). All of the cultivated species whose nitrogenase genes are included in this cluster, such as sulfate reducers and clostridia, are strict anaerobes.
Recently, nitrogenase gene sequences included in this cluster have been recovered from anoxic environments (including marine sediments and soils [33, 42]) and, most interestingly, from termite guts (25). The nitrogen-fixing species associated with zooplankton appear to be largely anaerobic microorganisms, which is consistent with the postulated existence of such organisms in the zooplankton gut (27). Therefore, since the nifH sequences obtained from zooplankton were largely representative of anaerobic microbes, the nitrogen-fixing microorganisms associated with zooplankton were probably derived from the gut rather than associated with the exoskeleton.
The intriguing finding that nitrogen fixation in the open ocean may occur in marine invertebrate guts is analogous to nitrogen fixation in guts of terrestrial insects (25) and marine shipworms (4). Sequences of this cluster are not found in the bulk water samples, indicating that the organisms from which these sequences were derived may be permanent residents of the zooplankton gut and may even be symbiotic with zooplankton. Planktonic invertebrates, including copepods, are known to have gut microflora (24), but this is the first report that demonstrates that copepod gut microflora includes diverse populations of microorganisms with the genetic potential for nitrogen fixation. Since nitrogen fixation is energetically expensive, zooplankton gut tracts may be an important environment for nitrogen fixation in oligotrophic oceans. Copepods are the most abundant zooplankton in the ocean (19), and since copepods are responsible for consuming much of the phytoplankton productivity in the world's oceans, they would provide the microflora with a continuous supply of energy-rich substrates for microbial metabolism. Anoxic conditions within the gut could protect nitrogenase from oxygen inactivation. Furthermore, it has recently been argued that nitrogen fixation in the ocean is limited by iron availability (11), but copepod gut tracts undergo pH (26) and redox changes during feeding and digestion that could be important in increasing the bioavailability of trace elements, such as iron, for nitrogen fixers in the gut tract. Copepod grazing clearly solubilizes phytoplankton cellular iron (15), which provides a potential mechanism for making iron available to copepod-associated nitrogen-fixing gut microflora.
The results of this study show that there are diverse nitrogen-fixing phylotypes in the oceanic environment in the plankton, as well as associated with diatom aggregates and planktonic crustacea. It has yet to be demonstrated whether these phylotypes fix nitrogen or how abundant these phylotypes are in the ocean. Recently, an approach for quantifying nifH genes was applied to the estuarine environment and showed that nif gene abundance decreased from freshwater sources to the mesohaline reaches (1). In comparison to results from the estuarine study, the amplification products from marine samples indicate that the concentration of nitrogen-fixing organisms is much lower in oceanic environments than in coastal environments. However, even at low densities, active populations of nitrogen-fixing microorganisms over vast areas of the open ocean could contribute substantially to nitrogen inputs in the world's oceans. This study provides the first evidence of the presence and diversity of open-ocean diazotrophs, which may be key to balancing the nitrogen budget of the oceans (22). The results indicate that there are far more diverse nitrogen-fixing microorganisms in the oceanic plankton than previously believed.
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ACKNOWLEDGMENTS |
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We thank Bermuda Biological Station personnel for sample collection and logistic support at the BATS station collections, and we thank D. Karl and L. Tupas for facilitating sample collection at the HOT station. We also thank D. Capone and T. Villareal for providing equatorial Atlantic Ocean picoplankton and Pacific Ocean diatom samples, respectively. L. Proctor provided copepod samples from the Gulf of Mexico and reviewed the manuscript.
This research was supported by NSF grants OCE-950353 and IBN 9629314 to J.P.Z.
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FOOTNOTES |
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* Corresponding author. Mailing address: Dept. of Biology, MRC 303, Rensselaer Polytechnic Institute, Troy, NY 12180-3590. Phone: (518) 276-8386. Fax: (518) 276-2162. E-mail: zehrj{at}rpi.edu.
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Abstract
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Materials & Methods
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Applied and Environmental Microbiology, September 1998, p. 3444-3450, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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[Abstract] [Full Text] -
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