Abstract
A geographically isolated maize landrace cultivated on nitrogen-depleted fields without synthetic fertilizer in the Sierra Mixe region of Oaxaca, Mexico utilizes nitrogen derived from the atmosphere and develops an extensive network of mucilage-secreting aerial roots that harbors a diazotrophic (N2-fixing) microbiota. Targeting these diazotrophs, we selected nearly 600 microbes of a collection obtained from mucilage and confirmed their ability to incorporate heavy nitrogen (15N2) metabolites in vitro. Sequencing their genomes and conducting comparative bioinformatic analyses showed that these genomes had substantial phylogenetic diversity. We examined each diazotroph genome for the presence of nif genes essential to nitrogen fixation (nifHDKENB) and carbohydrate utilization genes relevant to the mucilage polysaccharide digestion. These analyses identified diazotrophs that possessed the canonical nif gene operons, as well as many other operon configurations with concomitant fixation and release of >700 different 15N labeled metabolites. We further demonstrated that many diazotrophs possessed alternative nif gene operons and confirmed their genomic potential to derive chemical energy from mucilage polysaccharide to fuel nitrogen fixation. These results confirm that some diazotrophic bacteria associated with Sierra Mixe maize were capable of incorporating atmospheric nitrogen into their small molecule extracellular metabolites through multiple nif gene configurations while others were able to fix nitrogen without the canonical (nifHDKENB) genes.
Highlights
Nitrogen is an essential macroelement for plant productivity that is often limiting to plant growth when the natural abundance of its bio-available forms is depleted in the environment
To elucidate the genomic determinants for biological nitrogen fixation (BNF) by mucilagederived diazotrophs, we examined their genomes for the presence of carbohydrate active enzymes (CAZymes) and sugar transporters related to mucilage polysaccharide utilization, the canonical nif genes based on the Dos Santos model [7] with the Klebsiella pneumoniae NIF regulon as the model framework, and known alternative nif genes
We investigated the genomic mechanism for the diazotrophic phenotype (i.e. BNF) by examining the predicted coding sequences using Hidden Markov Models (HMMs) for the six nif genes of the Dos Santos model [7] within the context of seven genetic operons comprising the K. pneumoniae NIF regulon, which included: 1) the operon of nif genes involved in regulation of the nif pathway, nifRLA; 2) the catalytic operon, nifHDK; 3) operons involved in formation of the functional Fe-Mo protein, nifEN and nifBQ; 4) an operon of genes involved in assembly of the functional enzyme complex, nifUSVM; and 4) operons conferring genes associated with mediating electron transfer, nifJ and nifWF [43, 44]
Summary
Nitrogen is an essential macroelement for plant productivity that is often limiting to plant growth when the natural abundance of its bio-available forms is depleted in the environment. Exogenous nitrogen is currently provided for maize cultivation either through synthetic Haber-Bosch fertilizer produced at high environmental and economic cost [1], or from crop rotation with legumes that replenish field nitrogen levels by symbiotic association with. Bacteria from maize mucilage fix nitrogen with and without nif genes subject to the full terms and obligations of the ABS agreement and the authorization from the government of Mexico. Individuals wishing to access nucleic acid sequence data for scientific research activities should contact Mars Incorporated Chief Science Officer at CSO@effem. Individuals wishing to access nucleic acid sequence data for scientific research activities should contact Mars Incorporated Chief Science Officer at CSO@effem. com
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