Experiments linking nitrogenase gene expression to nitrogen fixation in the North Pacific subtropical gyre

We examined diazotroph activity in controlled experiments containing natural populations of nitrogen (N2)fixing microbes collected offshore from Kane’ohe Bay, Hawai’i, and from Station ALOHA (A Long-term Oligotrophic Habitat Assessment) in the North Pacific subtropical gyre. Quantitative polymerase chain reaction and reverse transcription-polymerase chain reaction approaches showed that the abundances of the major phylotypes did not change substantially during experimental incubations. Diel phasing of nifH gene expression in the experiments was similar to that documented for natural populations. Cell-specific N2 fixation rates, calculated as N2 incorporation rate divided by specific phylotype abundance (gene copy number), varied between the experiments, but generally agreed with the abundance and levels of nifH expression of each group of diazotrophs. Phosphorus additions had little effect on the abundance, gene expression, or N2 fixation activity of diazotrophs across the course of the 36-h experiments, indicating that other factors may limit the growth, gene expression, and activity of diazotrophs in the North Pacific subtropical gyre. Oceanic biological nitrogen (N2) fixation (BNF) has received increasing attention because of its important role in controlling the availability of nitrogen (N) for primary productivity and carbon sequestration (Gruber 2005). Oceanic BNF is controlled by several groups of cyanobacteria and possibly other noncyanobacterial prokaryotes (Zehr et al. 2001; Montoya et al. 2004; Church et al. 2005a). However, the contribution of individual diazotroph groups and the factors that constrain the growth of N2-fixing microorganisms and N2 fixation rates in the oceans are not well known. Redfield’s analysis of ocean elemental stoichiometry suggested that imbalances in the availability of nitrogen (N) and phosphorus (P) relative to the needs of phytoplankton would select for N2-fixing microorganisms (Redfield 1958), which was echoed by the idea of a nitrogen homeostat (‘‘nitrostat’’) in a modeling analysis of the N cycle of the global ocean (Tyrrell 1999). Conversely, Klausmeier et al. (2004) note that there was significant variability in N : P ratios that is species-specific and is affected by physiological state and growth rate. Resolving this controversy and understanding the factors that limit and select for diazotrophs is critical for understanding the dynamics of the N cycle in the global ocean. P and/or iron (Fe) are essential nutrients, the availability of which might control the growth of diazotrophs ( SañudoWilhelmy et al. 2001; Karl 2002). In the North Pacific subtropical gyre, increased rates of N2 fixation are hypothesized to drive the system toward P limitation, even though the in situ concentrations of inorganic P are higher than in some P limited systems (Karl et al. 2001). There may be colimitation by Fe and P, and the interactions and relative availability of these nutrients may be highly dynamic, with short-term and long-term changes that affect N2-fixing microorganisms. The limitation of N2 fixation by P may depend on the characteristics of the specific diazotroph, including uptake capabilities and cell size. There are several groups of nifH-containing microorganisms, including two distinct groups of unicellular cyanobacterial nifH sequences (termed Groups A and B) present at Station (Sta.) ALOHA (A Long-term Oligotrophic Habitat Assessment) of the Hawai’i Ocean Time-Series (HOT) program station near Oahu (Zehr et al. 1998; Zehr 1 Corresponding author ([email protected]).