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

Hanna Farnelid,Stefan Bertilsson,Grieg F Steward,Anders F Andersson,Søren Sørensen,Lars H Hansen,Lasse Riemann,Waleed Abu Al-Soud,Åke Hagström,Jack Anthony Gilbert

doi:10.1371/journal.pone.0019223

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.

Highlights

The availability of nitrogen (N) limits biological production in vast areas of the global ocean and is tightly linked to the fixation of atmospheric carbon dioxide and export of carbon from the ocean’s surface [1]
The pyrosequencing chemistry used here only allowed for retrieval of partial nifH genes; phylogenetic trees based on short fragments from reference nifH genes (60 amino acids) compared to the length usually used for nifH phylogeny (108 amino acids) showed only few topological differences (Figure S3) and sequences from cultivated representatives clustered within previously designated nifH Clusters (Figures 2 and S4)
Cluster I includes mainly nifH sequences from Cyanobacteria, Alpha, Beta- and Gammaproteobacteria, Cluster III includes anaerobes, such as methanogens, Clostridia, and some Deltaproteobacteria, while Cluster IV includes diverse nifH homologs found in methanogens [4]

Summary

Introduction

The availability of nitrogen (N) limits biological production in vast areas of the global ocean and is tightly linked to the fixation of atmospheric carbon dioxide and export of carbon from the ocean’s surface [1]. A proper understanding of the marine N cycle is needed for accurate quantification and forecasting of oceanic carbon cycling. Using best current estimates for each process, the calculated global oceanic N budget is out of balance with losses exceeding inputs [2], possibly as a consequence of incomplete knowledge about the identity, diversity, and autecology of N2-fixing microorganisms (diazotrophs; [3]). It is pertinent to address the composition and ecological dynamics of diazotrophs in the global ocean in order to understand, and predict, ecosystem productivity and carbon dynamics.

Methods

Results

Conclusion