Nitrogenase Gene Amplicons from Global Marine Surface Waters Are Dominated by Genes of Non-Cyanobacteria

Abstract

Cyanobacteria are thought to be the main N2-fixing organisms (diazotrophs) in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our analysis of 79,090 nitrogenase (nifH) PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples) collected at ten marine locations world-wide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III) were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least occasionally expressed. The contribution of non-cyanobacterial diazotrophs to the global N2 fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant.

Climate relevant trace gases (N2O and CH4) in the Eurasian Basin (Arctic Ocean)

“…Also, the diazotrophic community may be dominated by heterotrophic bacteria (Farnelid et al, 2011(Farnelid et al, , 2013Moisander et al, 2014;Bentzon-Tilia et al, 2015). In this study, significant N 2 and N 2 O fixation rates were determined in surface waters and in ice brine during the same expedition cruise.…”

Active nitrogen-fixing heterotrophic bacteria at and below the chemocline of the central Baltic Sea

“…As the cDNA samples from 41, 132 and 233 m yielded faint PCR products they were first amplified with nifH1 and nifH2 primers (Zehr and McReynolds, 1989) followed by 10 PCR cycles with barcoded primers (Farnelid et al, 2011). Nontranscribed RNA samples were included as control for incomplete DNA digestion.…”

“…These were then trimmed, sequences were clipped at 219 bp, translated into amino acids (73 aa), and sequences having in-frame stop codon(s) were removed. Unique reads were aligned as previously described (Farnelid et al, 2011). Reads were screened to check for homology to all reported nifH contaminant sequences, but no identical reads were found.…”

High levels of heterogeneity in diazotroph diversity and activity within a putative hotspot for marine nitrogen fixation

“…Ammonia ( primers under the same PCR reaction conditions (Dowd et al, 2008;Farnelid et al, 2011Farnelid et al, , 2013Messer et al, 2015;Supplementary Information). Raw sequences were quality filtered, whereby sequences with a quality score o25 and reads o200-bp long were removed and clustered into operational taxonomic units (OTUs) at 95% sequence identity (Penton et al, 2013) using UCLUST (Edgar, 2010) and rarefied to the lowest number of sequences per sample (872 sequences) in QIIME (Caporaso et al, 2010).…”