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The importance of inorganic N (NH₄⁺ or NO₃) for the nutrition and growth of marine phytoplankton has long been recognized (5, 7, 8), while the utilization of inorganic N by bacteria has historically received less attention (11, 13, 15, 17, 43). The primary role of heterotrophic bacteria is classically considered to be the decomposition and mineralization of dissolved and particulate organic nitrogen (27). Bacterial NO₃ assimilation is not a pathway currently considered in pelagic carbon and nitrogen cycle models (1, 6, 10). A recent review of freshwater and marine studies, however, reported that bacteria may rely on both NH₄⁺ and NO₃ for growth and biomass synthesis, and overall they may be significant consumers of inorganic N; mean consumption values of 30 and 40% have been reported for NH₄⁺ and NO₃, respectively (14). Under certain conditions, such as in the presence of high concentrations of dissolved organic carbon relative to the concentration of dissolved organic nitrogen, bacteria may be responsible for most, if not all, of the observed NO₃ uptake and disappearance (16, 24, 25). Significant heterotrophic bacterial utilization of dissolved inorganic nitrogen likely would have profound effects on the fluxes of N and C in the water column.

Bacterial nitrate utilization in aquatic communities, however, is difficult to study by conventional tracer approaches. Within the bacterial size class, autotrophic cyanobacteria (picoplankton) are often abundant (4, 41) and are likely to complicate conclusions regarding the total flux of labeled nitrogen into the heterotrophic fraction of the bacterial community. Also, size fractionation does not allow for examination of nitrate uptake by attached bacteria or large cells caught in filters.

It is known that some, but not all, heterotrophic bacteria are capable of growth on NO₃ as a sole N source (28). In Klebsiella pneumoniae, the structural genes for nitrate assimilation form an operon, nasFEDCBA (19-21). The NASC protein is thought to mediate electron transfer from NADH to NASA, which contains the active site for nitrate reduction (19, 20). The NASA protein has also been purified from a phototrophic member of the alpha subclass of the class Proteobacteria, Rhodobacter capsulatus, and characterized (2, 22). Examination of currently available prokaryotic genome sequences suggests that nasA is present in a wide diversity of organisms, although these observations need verification (28).

Molecular techniques can be employed to illuminate factors which control the rates of fluxes and transformations in nitrogen-cycling processes (40, 46, 47, 51). Molecular approaches have been successfully used to detect and characterize bacteria and the genes that are important in several aspects of the nitrogen cycle, including nitrification, dentrification, and nitrogen fixation (18, 29, 30, 37-39, 45, 48-50).

Here we describe the design and optimization of a series of nested heterotrophic bacterium-specific nasA PCR primers. The detection of nasA genes in a variety of marine environments provided a basis for the hypothesis that the potential for NO₃ utilization by heterotrophic bacteria is significant. Phylogenetic analysis of nasA genes cloned from diverse samples indicated that there are several distinct clades and suggests that there is a clear genetic distinction between nasA genes from heterotrophic bacteria and nasA genes from autotrophic cyanobacteria.

Initially, three nested universal degenerate nasA primers were designed based on five previously determined sequences from cyanobacteria and one sequence from a heterotrophic bacterium (3, 12, 20, 23, 34). The sequences from cyanobacterial strains were from Oscillatoria chalybea, Anabaena sp. strain PCC7120, Synechocystis sp. strain PCC6803, a Synechococcus sp., and Synechococcus sp. strain 7942, and the sequence from a heterotrophic strain was from Klebsiella oxytoca. The GenBank accession numbers for these sequences are X89445, L49163, BAA17488, CAA52675, P39458, and L06800, respectively. An alignment of the inferred amino acid sequences encoded by nasA indicated that there were conserved regions suitable for targeting by PCR oligonucleotide primers. Such primers have been used to amplify nasA sequences in other heterotrophic bacteria, and a group-specific degenerate primer was designed to specifically amplify the nasA gene from heterotrophic bacteria. All of the primers used in this study are listed in Table 1.

Surface water samples were collected during two cruises in the South Atlantic Bight (SAB) off the Georgia coast aboard the R/V Bluefin during October 1998 and aboard the R/V Cape Hatteras during March 1999 (31 to 33°N, 78 to 81°W). SAB samples were also collected from docks located on the Skidaway River (March 1999) and the Wilmington River (July 1998). Additional water samples used in this study were collected at depths of 5, 30, and 80 m in the Barents Sea (70 to 78°N, 30°E) aboard the R/V Jan Mayen during July 1999 and from the surface of the North Pacific Gyre at Hawaii Ocean Time Series stations (22°45'N, 158°W) during May 1997 and aboard the R/V Melville during June 1999. For DNA extraction, bacteria were collected from 40 liters of water. To remove eukaryotic plankton, the water was prefiltered under a vacuum through a 3-µm-pore-size polycarbonate cartridge filter (Gelman Sciences, Inc., Ann Arbor, Mich.) and then through a 142-mm-diameter, 0.8-µm-pore-size polycarbonate Supor filter (Gelman) with a custom-manufactured acrylic filter holder. Bacterial cells in the filtrate were collected on a 142-mm-diameter, 0.2-µm-pore-size polycarbonate Supor filter (Gelman) and stored at 20°C aboard ship and then transferred to storage at 80°C in the lab. DNA in the SAB samples collected in October 1998 was extracted as described by Gonzalez et al. (9), and DNA in all other samples was extracted with an UltraClean Mega Prep soil DNA kit (Mo Bio Laboratories, Inc., Solana Beach, Calif.). For the latter procedure, frozen filters were crushed inside Whirl-Pak bags (Nasco, Fort Atkinson, Wis.) and put directly into the lysing matrix used for step one of the soil DNA extraction procedure. Visualization of purified DNA by gel electrophoresis revealed the presence of high-molecular-weight DNA with little shearing and no RNA contamination. From 40 liters of seawater, this method typically yielded an average of 100 to 110 µg of DNA. If it was assumed that the average concentration bacterial cells was 10⁶ cells per ml of seawater and the average DNA content was 3 fg/cell (31), the approximate extraction efficiency of this method was between 80 and 90%.

PCR was performed by using a nested format to improve specificity and sensitivity. The PCR products obtained with the outermost degenerate nasA/narB universal primers (nas22, nas1933) were subsequently used as templates in PCR with the heterotroph-specific internal primer set (nas964, nasA1735). Amplification was accomplished by using the Qiagen Taq PCR Master Mix System and the standard protocol recommended by the vendor (Qiagen, Valencia, Calif.); a hot start at 94°C for 5 min was followed by 35 cycles consisting of 94°C for 5 s, 55.5°C for 10 s, and 72°C for 1 min, with a 7-min final extension step at 72°C. DNA template (10 to 100 ng of community DNA or 0.1 to 10 ng of genomic DNA from a pure culture) was added to each 25-µl PCR mixture. First-round reaction mixtures contained 1 µM primer nas22, 1 µM primer nas1933, and 3.5 mM MgCl₂. Second-round nested PCR mixtures contained 1 to 2 µl of product from the first round, 2.5 mM MgCl₂, 1 µM primer nas964, and 1 µM primer nasA1735, and the extension time in each cycle was decreased to 30 s. The nasA-specific primers yielded a PCR product that was 750 to 800 bp long. 16S rRNA amplification of the nearly complete 16S rRNA gene was facilitated by using eubacterial primers fd1 and rp2 (Table 1) (42) (100 nM each). Thermal cycling was performed with a model 2400 or 9700 thermal cycler (Perkin-Elmer Corp., Norwalk, Conn.).

Although nasA PCR could be optimized for specific community DNA samples by raising the annealing temperature to 57 to 60°C, a somewhat less stringent annealing temperature, 55°C, was used during the initial construction of clone libraries to increase the yield and the likelihood of amplification with most primer-template combinations.

The PCR product of the desired size was excised from the gel and purified by using GenElute agarose spin columns (Supelco, Bellefonte, Pa.). PCR products were ligated and cloned by using either a TOPO TA Cloning kit (for pure cultures) or an Original TA Cloning kit (for community DNA samples). In both cases, the PCR product was ligated into a pCR 2.1 plasmid vector and cloned into TOP10 One Shot competent Escherichia coli cells (Invitrogen, Carlsbad, Calif.). Plasmid DNA was extracted and purified by using the Wizard Plus Minipreps DNA purification system (Promega, Madison, Wis.).

Sequences were determined by automated sequencing at the Molecular Genetics Facility (University of Georgia) with ABI automated sequencers (models 373 and 377). Sequencing reactions were facilitated by using an ABI Big Dye Prism dideoxy sequencing dye terminator kit as recommended by the manufacturer. Sequence analysis was accomplished by using ABI software, version 3.3 (ABI, Foster City, Calif.). The sequencing primers used are listed in Table 1.

Bacteria were isolated from seawater samples collected from the SAB continental shelf during March and June 1999 (31 to 33°N, 78 to 81°W). Bacteria were also isolated from Barents Sea water samples collected during July 1999 (70 to 80°N, 30°E). Bacteria were isolated by using either organic nitrogen or nitrate as the sole nitrogen source. Selected colonies were axenically transferred to new plates twice to ensure that pure cultures were obtained. For long-term storage each isolate was maintained in a 15% (vol/vol) glycerol freezer stock preparation at 80°C.

To screen isolated strains for the presence of nasA, PCR-amenable DNA was extracted from each of the isolates by using the FastDNA spin protocol and a Fast Prep instrument (both from BIO 101, Vista, Calif.). In all PCRs, appropriate negative controls without DNA and positive controls were included.

To test isolates for the ability to grow on nitrate as the sole N source, two tubes containing 5 ml of NFG medium (33) were prepared. To one of the tubes a sterile NaNO₃ solution was added to obtain a final NO₃ concentration of 10 mM. The second tube did not receive such an addition and served as a negative control. The two NFG medium tubes were then inoculated 1:100 with a stationary-phase culture grown in peptone- and yeast-enriched artificial seawater (26). After 72 h, the optical densities of the two tubes were compared to the optical density of a NFG medium tube that had not been inoculated. Additionally, several isolates were selected for batch culture growth assays. These experiments were conducted in axenic 100-ml NFG medium cultures containing 80 µM or 10 mM NO₃ as the sole N source. Doubling rates were determined by estimating cell density at a minimum of four time points during the exponential growth phase. Cell densities were determined by direct epiflourescent microscopy after staining with DAPI (4',6'-diamidino-2-phenylindole) (44).

Phylogenetic relationships based on nasA gene sequences were determined. Nucleotide sequences were translated into approximately 264 unambiguous amino acids. All of the available narB/nasA amino acid sequences were then aligned by using the CLUSTAL W (version 1.7) multiple-sequence-alignment algorithm (32). Phylogenetic trees were inferred and drawn by using the TREECON software package (version 1.3b) (35, 36) and the Kimura two-parameter model for inferring evolutionary distances. Bootstrap estimates (100 replicates) of confidence intervals were also made by using the algorithms available in the TREECON package.

For 16S rRNA analysis, 464 unambiguously alignable nucleotide positions were used. The nucleotide sequences were compared to 16S rRNA gene sequences available in the GenBank database by using the Blast program to determine the degrees of sequence similarity to known organisms. All of the nasA and 16S rRNA sequences determined in this study (Table 2) have been deposited in the GenBank database.

Results. Using the universal nasA nested primer set, we amplified, cloned, and sequenced a 1,000-bp fragment from a group of phylogenetically diverse bacteria, including Clostridium oceanica, Vibrio diazotrophicus, a Pseudomonas sp., Trichodesmium sp. strain IMS101, a Fischerella sp., and Plectonema boryanum, as well as from DNA extracted from the bacterial size fraction of seawater collected at a Hawaii Ocean Times Series station near Hawaii. Also, we attempted to amplify the 1,000-bp nasA fragment from Bacillus sp., Micrococcus luteus, Vibrio sp. strain S-14, and Pseudomonas stutzeri. These templates, however, did not yield a PCR product, and we concluded that they were nasA negative (Table 2).

View larger version (86K): [in this window] [in a new window]   FIG. 1.   PCR amplification of the expected 750- to 800-bp nasA gene fragment from various marine samples. Lane 2 contained a PCR product from a sample that was prepared by amending filtered seawater with 103 K. pneumoniae cells/ml (final concentration). nasA PCR products were amplified from samples collected from a Norwegian fjord (lane 3), the Barents Sea (lanes 4 to 6), the Skidaway River (lanes 7 and 8), the SAB (lanes 9 and 10), and the North Pacific Gyre (lanes 11 and 12). Lane 1 contained a molecular weight standard PCR marker (Promega).

View larger version (37K): [in this window] [in a new window]   FIG. 2.   Inferred phylogenetic relationships of nasA- and narB-encoded amino acid sequences from heterotrophic bacteria and cyanobacteria, respectively. The scale bar indicates 0.05 fixed amino acid substitution per site. The numbers at the nodes are bootstrap values. Bootstrap values less than 70 (of 100) are not shown. The amino acid sequence of formate dehydrogenase from Methanobacterium thermoautotrophicum (GenBank accession number U52681), a putative evolutionary ancestor of the proteins encoded by the nasA and narB genes, was used to root the tree. HOTS, Hawaii Ocean Time Series.

Conclusions. The correlation between the presence of nasA and nitrate utilization assay results for individual isolates (34 of 36 isolates tested) supports the hypothesis that the nasA-specific primer sets developed in this study provide a reliable assay for functional assimilatory nitrate reductase genes. Sequences derived from isolates that were unable to utilize NO₃ in culture (Table 3) are more closely related to dehydrogenases and proteins encoded by members of other gene families and can be distinguished phylogenetically from functional assimilatory nitrate reductases (Fig. 2). This illustrates the fact that although degenerate primers are powerful and able to retrieve gene fragments from very diverse organisms, it is important to sequence and phylogenetically analyze PCR products from as many different types of organisms as possible in order to identify potential nonspecific PCR products.

Nucleotide sequence accession numbers. The nasA and 16S rRNA sequences determined in this study have been deposited in the GenBank database under the accession numbers shown in Table 2 and Fig. 2.