SummaryBiological nitrogen fixation is an important source of fixed nitrogen for the biosphere. Microorganisms catalyse biological nitrogen fixation with the enzyme nitrogenase, which has been highly conserved through evolution. Cloning and sequencing of one of the nitrogenase structural genes, nifH , has provided a large, rapidly expanding database of sequences from diverse terrestrial and aquatic environments. Comparison of nifH phylogenies to ribosomal RNA phylogenies from cultivated microorganisms shows little conclusive evidence of lateral gene transfer. Sequence diversity far outstrips representation by cultivated representatives. The phylogeny of nitrogenase includes branches that represent phylotypic groupings based on ribosomal RNA phylogeny, but also includes paralogous clades including the alternative, non-molybdenum, non-vanadium containing nitrogenases. Only a few alternative or archaeal nitrogenase sequences have as yet been obtained from the environment. Extensive analysis of the distribution of nifH phylotypes among habitats indicates that there are characteristic patterns of nitrogen fixing microorganisms in termite guts, sediment and soil environments, estuaries and salt marshes, and oligotrophic oceans. The distribution of nitrogen-fixing microorganisms, although not entirely dictated by the nitrogen availability in the environment, is nonrandom and can be predicted on the basis of habitat characteristics. The ability to assay for gene expression and investigate genome arrangements provides the promise of new tools for interrogating natural populations of diazotrophs. The broad analysis of nitrogenase genes provides a basis for developing molecular assays and bioinformatics approaches for the study of nitrogen fixation in the environment.

Fixed nitrogen (N) often limits the growth of organisms in terrestrial and aquatic biomes, and N availability has been important in controlling the CO2 balance of modern and ancient oceans. The fixation of atmospheric dinitrogen gas (N2) to ammonia is catalysed by nitrogenase and provides a fixed N for N-limited environments. The filamentous cyanobacterium Trichodesmium has been assumed to be the predominant oceanic N2-fixing microorganism since the discovery of N2 fixation in Trichodesmium in 1961 (ref. 6). Attention has recently focused on oceanic N2 fixation because nitrogen availability is generally limiting in many oceans, and attempts to constrain the global atmosphere-ocean fluxes of CO2 are based on basin-scale N balances. Biogeochemical studies and models have suggested that total N2-fixation rates may be substantially greater than previously believed but cannot be reconciled with observed Trichodesmium abundances. It is curious that there are so few known N2-fixing microorganisms in oligotrophic oceans when it is clearly ecologically advantageous. Here we show that there are unicellular cyanobacteria in the open ocean that are expressing nitrogenase, and are abundant enough to potentially have a significant role in N dynamics.

This paper presents standards and best practices for reporting genome sequences of uncultivated viruses.Supplementary informationThe online version of this article (doi:10.1038/nbt.4306) contains supplementary material, which is available to authorized users.

Bacterial community composition, enzymatic activities, and carbon dynamics were examined during diatom blooms in four 200-liter laboratory seawater mesocosms. The objective was to determine whether the dramatic shifts in growth rates and ectoenzyme activities, which are commonly observed during the course of phytoplankton blooms and their subsequent demise, could result from shifts in bacterial community composition. Nutrient enrichment of metazoan-free seawater resulted in diatom blooms dominated by a Thalassiosira sp., which peaked 9 days after enrichment (Ϸ24 g of chlorophyll a liter ؊1 ). At this time bacterial abundance abruptly decreased from 2.8 ؋ 10 6 to 0.75 ؋ 10 6 ml ؊1, and an analysis of bacterial community composition, by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments, revealed the disappearance of three dominant phylotypes. Increased viral and flagellate abundances suggested that both lysis and grazing could have played a role in the observed phylotype-specific mortality. Subsequently, new phylotypes appeared and bacterial production, abundance, and enzyme activities shifted from being predominantly associated with the <1.0-m size fraction towards the >1.0-m size fraction, indicating a pronounced microbial colonization of particles. Sequencing of DGGE bands suggested that the observed rapid and extensive colonization of particulate matter was mainly by specialized ␣-Proteobacteria-and Cytophagalesrelated phylotypes. These particle-associated bacteria had high growth rates as well as high cell-specific aminopeptidase, ␤-glucosidase, and lipase activities. Rate measurements as well as bacterial population dynamics were almost identical among the mesocosms indicating that the observed bacterial community dynamics were systematic and repeatable responses to the manipulated conditions. During phytoplankton blooms in the ocean, primary productivity and its processing by the food web create a heterogeneous environment of particulate, colloidal, and dissolved organic matter in a continuum of size classes and concentrations (2,4,35,76). Major changes in organic matter concentration and composition are expected to occur at different stages of the bloom. The variations in the organic matter regime are typically accompanied by pronounced changes in bacterial abundance, productivity, ectohydrolase activities, and colonization of particles (48,68). In one recent mesocosm experiment (68), growth rates, colonization, and enzyme activities generally increased following the peak of a diatom bloom but different enzymes were found to peak at different times. In principle, these changes could have occurred without major shifts in the phylogenetic composition of the bacterial community. Shifts in activity and surface attachment would represent plasticity in the bacterial phenotypes, with enzyme expression and growth being regulated in response to the available organic substrates. Alternatively, the observed changes could have resulted from community succession, with bacteria with...