RESUMEN
BACKGROUND: The metabolic capacity for nitrogen fixation is known to be present in several prokaryotic species scattered across taxonomic groups. Experimental detection of nitrogen fixation in microbes requires species-specific conditions, making it difficult to obtain a comprehensive census of this trait. The recent and rapid increase in the availability of microbial genome sequences affords novel opportunities to re-examine the occurrence and distribution of nitrogen fixation genes. The current practice for computational prediction of nitrogen fixation is to use the presence of the nifH and/or nifD genes. RESULTS: Based on a careful comparison of the repertoire of nitrogen fixation genes in known diazotroph species we propose a new criterion for computational prediction of nitrogen fixation: the presence of a minimum set of six genes coding for structural and biosynthetic components, namely NifHDK and NifENB. Using this criterion, we conducted a comprehensive search in fully sequenced genomes and identified 149 diazotrophic species, including 82 known diazotrophs and 67 species not known to fix nitrogen. The taxonomic distribution of nitrogen fixation in Archaea was limited to the Euryarchaeota phylum; within the Bacteria domain we predict that nitrogen fixation occurs in 13 different phyla. Of these, seven phyla had not hitherto been known to contain species capable of nitrogen fixation. Our analyses also identified protein sequences that are similar to nitrogenase in organisms that do not meet the minimum-gene-set criteria. The existence of nitrogenase-like proteins lacking conserved co-factor ligands in both diazotrophs and non-diazotrophs suggests their potential for performing other, as yet unidentified, metabolic functions. CONCLUSIONS: Our predictions expand the known phylogenetic diversity of nitrogen fixation, and suggest that this trait may be much more common in nature than it is currently thought. The diverse phylogenetic distribution of nitrogenase-like proteins indicates potential new roles for anciently duplicated and divergent members of this group of enzymes.
Asunto(s)
Genoma Arqueal/genética , Genoma Bacteriano/genética , Fijación del Nitrógeno/genética , Nitrogenasa/genética , Secuencia de Aminoácidos , Archaea/clasificación , Archaea/enzimología , Archaea/genética , Proteínas Arqueales/química , Proteínas Arqueales/genética , Bacterias/clasificación , Bacterias/enzimología , Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Biodiversidad , Vías Biosintéticas/genética , Secuencia Conservada/genética , Genes Arqueales/genética , Genes Bacterianos/genética , Datos de Secuencia Molecular , Nitrogenasa/química , Nitrogenasa/metabolismo , Filogenia , Estándares de Referencia , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
With the transition from manual to robotic HTS in the last several years, assay optimization has become a significant bottleneck. Recent advances in robotic liquid handling have made it feasible to reduce assay optimization timelines with the application of statistically designed experiments. When implemented, they can efficiently optimize assays by rapidly identifying significant factors, complex interactions, and nonlinear responses. This article focuses on the use of statistically designed experiments in assay optimization.