Facets of diazotrophy in the OMZ off Peru revisited- what we couldn’t see from a single marker gene approach

Biological dinitrogen (N2) fixation is the pathway making the large pool of atmospheric N2available to marine life. Besides direct rate measurements, a common approach to explore the potential for N2fixation in the ocean is a mining based on molecular genetic methods targeting the key functional genenifH, coding for a subunit of the nitrogenase reductase. As novel sequencing and single cell techniques improved, our knowledge on the diversity of marine N2fixers grew exponentially. However, to date one aspect of N2fixation in the ocean is commonly left aside. This is the existence of two alternative types of nitrogenases, which are besides the Mo-Fe nitrogenaseNif, the Fe-Fe nitrogenaseAnf, and the V-Fe nitrogenaseVnf, which differ with regard to their metal co-enzymes, as well as regarding their operon structure and composition.nifH-based studies may thus be biased, and alternative nitrogenases could not be recovered. Here, we screened a set of 6 metagenomes and -transcriptomes from a sulfidic water patch from the oxygen minimum zone off Peru for genes involved in N2fixation. We identified genes related to all three nitrogenases, and generally increased diversity as compared to our previousnifH-based study from the same waters. While we could not confirm gene expression of the alternative nitrogenases from our transcriptomes, we detected additional diazotrophs involved in N2fixation. We suggest that alternative nitrogenases may not be used under conditions present in those waters, however, depending on trace metal limitation in the future they may become active.Significance statementThis study addresses the important process of N2fixation, based on a whole-metagenome and –transcriptome screening, reports an increased diversity of N2fixing microbes in the sulfidic shelf water off Peru, as compared to previous target-gene based studies from the same waters. In addition to a generally higher diversity, genes encoding for alternative nitrogenases, which were previously not subject of any study on N2fixation in oxygen minimum zones, were detected. The ecological meaning and evolutionary history of those alternative nitrogenases is subject of ongoing debates, however, their presence in OMZ waters would allow for N2fixation at extreme anoxia, which may become important in a future ocean challenged by progressive deoxygenation.