Nitrogen fixation and nitrogenase (nifH) expression in tropical waters of the eastern North Atlantic

Abstract

Expression of nifH in 28 surface water samples collected during fall 2007 from six stations in the vicinity of the Cape Verde Islands (north-east Atlantic) was examined using reverse transcription-polymerase chain reaction (RT-PCR)-based clone libraries and quantitative RT-PCR (RT-qPCR) analysis of seven diazotrophic phylotypes. Biological nitrogen fixation (BNF) rates and nutrient concentrations were determined for these stations, which were selected based on a range in surface chlorophyll concentrations to target a gradient of primary productivity. BNF rates greater than 6 nmolN l(-1) h(-1) were measured at two of the near-shore stations where high concentrations of Fe and PO(4)(3-) were also measured. Six hundred and five nifH transcripts were amplified by RT-PCR, of which 76% are described by six operational taxonomic units, including Trichodesmium and the uncultivated UCYN-A, and four non-cyanobacterial diazotrophs that clustered with uncultivated Proteobacteria. Although all five cyanobacterial phylotypes quantified in RT-qPCR assays were detected at different stations in this study, UCYN-A contributed most significantly to the pool of nifH transcripts in both coastal and oligotrophic waters. A comparison of results from RT-PCR clone libraries and RT-qPCR indicated that a γ-proteobacterial phylotype was preferentially amplified in clone libraries, which underscores the need to use caution interpreting clone-library-based nifH studies, especially when considering the importance of uncultivated proteobacterial diazotrophs.

Figure 1

Neighbor-joining tree of partial nifH cyanobacterial sequences obtained from Cape Verde transcripts. Branch lengths were computed using the Jukes–Cantor correction in the ARB software. Bootstrapping was performed in MEGA 4.0, and bootstraps greater than 70% (1000 replicates) are shown next to the branches. Cape Verde transcripts are marked according to the key to emphasize the number of sequences represented by each OTU. All Trichodesmium, UCYN-A, UCYN-B and γ-24774A11 transcript sequences are targeted by the qPCR assays used in this study. Sequences that are related, but not identical, to phylotypes that are targeted with qPCR assays are marked with a white arrow. Sequences that are suspected contaminants are underlined, and those that have been determined to be contaminants in nifH-based PCR studies are marked with an asterisk.

Figure 2

Relative contribution of individual diazotrophs to overall nifH transcript pools quantified in surface waters at each station as determined by RT-qPCR. Results from sample B4 were not included, as it was taken below the mix layer. The α-24809A06 phylotype was not detected in any sample. Map produced using http://www.aquarius.geomar.de/omc/make_map.html.

Figure 3

Diel expression of cyanobacterial and proteobacterial nifH gene expression determined using RT-qPCR at Stations C–F. Sample C5 has been omitted from these plots owing to discrepancies in T/S. Bars at the top of each graph indicate night time (black) and daytime (white). If nifH transcripts per l were measured as DNQs, they were conservatively estimated to be 1 nifH transcript per l.Tricho.—Trichodesmium; RR—Richelia associated with Rhizosolenia; HR—Richelia associated with Hemiaulus.

Figure 4

Comparison of results from RT-PCR and RT-qPCR for UCYN-A, UCYN-B, Trichodesmium and γ-24774A11 at each station. (a) Frequency of each phylotype occurring in the RT-PCR clone libraries normalized to the sum of frequencies for all four phylotypes. (b) Percent contribution of individual phylotypes to the total nifH transcripts per l detected via RT-qPCR at all stations.