We describe here the design and utilization of a 16S rDNA oligonucleotide, NITRO821R, which we used in the PCR to detect marine unicellular cyanobacterial diazotrophs. This primer recognizes all known cultured isolates and environmental sequences in a well-defined lineage (Fig. ), which suggests that it should have broad utility for detection of these organisms in the natural environment. Confirmation that the cyanobacterial symbionts of the marine diatom C. frauenfeldianum
can fix atmospheric dinitrogen is still required, however. This would require isolation of these cyanobacterial symbionts in axenic culture. Assuming that such organisms do fix nitrogen, then the NITRO821R primer shows excellent specificity for marine unicellular cyanobacterial diazotrophs, particularly in conjunction with the cyanobacterium- and plastid-specific primer CYA359F (19
), in PCRs (Fig. ), as shown in this study.
Several 16S rDNA sequences of freshwater unicellular cyanobacterial diazotrophs which represent the genera Gloeocapsa, Gloeothece, and Aphanothece (accession numbers AB067575 to AB067581 and AB119259) and which are in the UCYN2-fix lineage described here (data not shown) have recently been deposited in the GenBank database. These sequences are also targeted with complete identity by the NITRO821R oligonucleotide, which potentially broadens its utility to freshwater systems. However, care is needed when this primer is used to enumerate this lineage in freshwater environments, given that we have shown that sequences of members of the genus Planktothrix, with a 1-bp mismatch with the NITRO821R primer, are amplified in PCRs with the CYA359F-NITRO821R primer pair. Specific size fractionation of freshwater environmental samples to remove the filamentous Planktothrix spp. would be one route to circumvent such a problem.
The utility of this molecular approach for revealing the distribution of unicellular cyanobacterial diazotrophs in natural marine systems appears to be well demonstrated, however, by the data obtained along the AMBITION cruise transect. Although the relative abundance compared to the nondiazotrophic picocyanobacterial genera Prochlorococcus
, which were the dominant organisms at the southern and northern stations, respectively (G. Tarran unpublished data), was low, significant numbers of the UCYN2
-fix lineage were detectable, as determined by the semiquantitative analytical PCR developed in this study. Interestingly, the range of cell abundance observed along this single transect (7 × 103
± 1.6 × 102
to 3.8 × 105
± 8 × 104
) is very similar to the range of average cell counts of unicellular cyanobacteria in the 2.5- to 7-μm size class reported by Falcon et al. (10
) for the tropical North Atlantic and subtropical North Pacific. In that study the cell numbers in the two ocean systems were considerably different, and it was proposed that the higher Fe flux to the North Atlantic was one of the factors that were important in determining the abundance of these organisms. Although we have no data on Fe flux at the different stations along the AMBITION cruise transect, this ocean basin is known to have high and variable deposition of dust (24
), which results in pulsed iron addition in this region (14
). Certainly, the fact that markedly different relative abundance values for the UCYN2
-fix lineage were observed along this transect shows that the abundance of these organisms can vary considerably over relatively small spatial scales. Indeed, a comparison of a suite of environmental parameters obtained from the AMBITION cruise suggests that seawater temperature (Fig. ) accompanied by low surface SRP and nitrogen levels (generally <0.25 μM) is significant in shaping the conditions in which these organisms can proliferate. Temperature has recently been shown to be important in dictating the distribution of heterocystous and nonheterocystous cyanobacteria in oceanic systems (23
). In that study, differences in the temperature dependence of oxygen flux, respiration, and N2
fixation activity were shown to be critical in explaining how Trichodesmium
performs better than heterocystous species at higher temperatures, and it may be that some of these factors are important for unicellular nitrogen-fixing species too.
The construction of environmental clone libraries based on the CYA359F-NITRO821R primer pair allowed us to obtain direct insight into the diversity of the UCYN2
-fix lineage along the AMBITION cruise transect. The wide genetic diversity among the derived 16S rDNA sequences for the three stations analyzed was noticeable (the lowest level of identity was 93.6% for the 462-bp product), and this diversity was much greater than the diversity that has been found for the picocyanobacterial genera Prochlorococcus
), for which the lowest levels of identity between strains and environmental sequences were 96 to 97% (for sequences usually >1,000 bp long) in their respective lineages. How this translates to physiological diversity requires further culture isolation since several of the sequences, particularly those from station 4, had no closely related cultured counterparts. Within a station, particularly stations 2 and 11, the genetic diversity observed was generally lower, and interestingly, sequences from the same station formed discrete clusters or clades (Fig. ), suggesting the potential for ecotype adaptation of members of the UCYN2
-fix lineage in specific water columns. The utility of the CYA359F-NITRO821R primer pair for focusing directly on the UCYN2
-fix lineage is mirrored by data for the nifH
clone libraries, where the primers used are not targeted to a particular cyanobacterial lineage. Here, sequences closely related to the Trichodesmium
group dominated the libraries. Two sequences from station 2, however, were closely related to the nifH
sequence of Crocosphaera
sp. strain WH 8501, which was in good agreement with the 16S rDNA environmental sequence data from that station, where sequences closely related to this strain dominated. These sequences thus correspond to the group B nifH
sequences designated by Zehr et al. (34
), while the group A nifH
sequences reported in that study may be related to the deeply branching 16S rDNA clusters that include the station 4 clones.
Given the wide utility of the nifH
gene for investigating the diversity of marine nitrogen fixers (32
), it is noteworthy that we also obtained two environmental sequences (from stations 2 and 11) whose closest known relatives are nifH
sequences from Anabaena
sp. We found no previous reports of nifH
sequences from members of this genus for oligotrophic open-ocean marine environments. However, these sequences may well be equivalent to the sequence of the novel Anabaena
sp. described by Carpenter and Janson (8
), who reported the presence of a heterocystous cyanobacterium in the southwest Pacific Ocean and Arabian Sea at a low level (1 to 4 trichomes liter−1
The NITRO821R oligonucleotide and the analytical PCR developed here thus provide an excellent tool for rapid and sensitive screening of environmental samples to establish the presence and relative abundance of unicellular cyanobacterial diazotrophs, organisms that play important roles in oceanic new production and biogeochemistry. Use of the oligonucleotide in fluorescent in situ hybridization or quantitative PCR experiments, in which absolute quantitation of cell numbers can be obtained, or use in reverse transcription-PCR to determine the members of the community that are active in situ would further enhance its value.