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Mechanistic insights bidirectional extracellular electron transfer driving synchronous nitrogen fixation in Klebsiella variicola

ABSTRACT: Microorganisms can function as anode catalysts to achieve biological nitrogen fixation while simultaneously generating bioelectricity. To date, only a limited number of microorganisms are capable of performing both outward and inward extracellular electron transfer (EET) concurrently. However, the variations in nitrogen fixation performance and underlying mechanisms during bidirectional EET remain poorly understood. Here, we isolated a nitrogen-fixing bacterium, Klebsiella variicola C1, which exhibits the ability for bidirectional electron transfer. Three-dimensional fluorescence and cyclic voltammetry analyses uncovered distinct differences in biofilm compositions at the anode and cathode. Fluorescence quantitative PCR and comparative proteomics highlighted specific distinctions in key genes and proteins associated with outward and inward EET processes. Outer-membrane lipoprotein carrier proteins likely play a primary role in outward electron transfer, whereas pilus proteins may predominantly facilitate inwa

ORGANISM(S): Klebsiella Variicola Uci 18

SUBMITTER: Peng Li

PROVIDER: PXD071420 | iProX | Mon Dec 01 00:00:00 GMT 2025

REPOSITORIES: iProX

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Project description:Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCE Azotobacter vinelandii is a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineered A. vinelandii strains and nitrogenase variants can be modulated at the regulatory level. The engineered strain studied here responds by adjusting the expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts.

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Mechanistic insights bidirectional extracellular electron transfer driving synchronous nitrogen fixation in Klebsiella variicola

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