Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

Abstract

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.

Keywords: Azotobacter vinelandii; RNA-Seq; ancestral protein reconstruction; nitrogen fixation; nitrogenase.

Fig 1

Construction and physiology of A. vinelandii strain ancNif harboring an ancestral nitrogenase NifD protein subunit, previously reported by Garcia et al. (20). (A) The protein sequence of ancestral NifD was inferred from a NifHDK protein phylogeny (the targeted ancestral NifD clade, which includes the A. vinelandii lineage, shown in bold). The ancestral nifD gene (pink) was integrated into the A. vinelandii genome by homologous recombination, replacing a kanamycin resistance marker (KanR) previously incorporated to knock out WT nifD (see Materials and Methods). The engineered ancestral gene is the only genetic perturbation within the broader nif major and minor clusters. (B) ColabFold-predicted structure of the hybrid nitrogenase catalytic tetramer, NifDK, in ancNif, generated in the present study. NifD subunits are colored tan, with residues within the ancestral NifD that are substituted relative to WT highlighted pink. NifK is shown as transparent. FeMo-co serves as both the site of N₂ reduction to NH₃, as well as reduction in the alternative substrate C₂H₂ to C₂H₄ (dotted arrow). (C) Growth curve and growth parameters of ancNif and WT. Midpoint time represents the time to the inflection point of a logistic curve fit to the growth data (26), which highlights the extended growth lag in ancNif. Average growth parameter values are tabulated (five biological replicates per strain) ±1 SD. (D) Acetylene reduction rates of ancNif and WT. The bar plot shows mean acetylene reduction rates (three biological replicates per strain) and error bars represent ±1 SD. (C-D) Asterisks indicate P < 0.05 relative to WT (one-way ANOVA, post-hoc Tukey HSD). (A–D). Figures modified from Garcia et al. (20).

Fig 2

Global differential gene expression in strain ancNif relative to WT. (A) Volcano plot highlighting significantly differentially expressed genes in ancNif vs WT, defined by an adjusted P < 0.05. Data points corresponding to nif major or minor cluster genes are indicated in dark blue (see Fig. 3). (B) Clustering of gene expression patterns across three biological replicates of ancNif.

Fig 3

Transcription levels across the (A) nif major and (B) nif minor clusters of ancNif and WT A. vinelandii strains, expressed as fragments per kilobase per million mapped reads (FPKM). Bars represent mean values across three biological replicates per strain, and error bars indicate ±1 SD. Asterisks indicate adjusted P < 0.05. Gene and transcriptional unit annotations from Del Campo et al. (10), the latter which mirrors operon predictions based on the transcriptional data presented here (Supplemental file 4).

Fig 4

Differentially expressed genes encoding proteins involved in cellular functions external to the immediate nitrogen fixation network in ancNif. (A) Transcript levels across representative clusters related to respiration, motility, stress response, and molybdate transport. Bars represent mean values across three biological replicates per strain, and error bars indicate ±1 SD. Adjusted P < 0.05 for all genes except those labeled as not significant (“n.s.”). Schematic illustrates the relevance of cellular functions to nitrogen fixation in A. vinelandii. Arrows next to each cellular component signify associated gene expression changes relative to WT, with arrow thickness roughly corresponding to the magnitude of change and arrow direction indicating either increased expression in ancNif (arrow pointing upward) or decreased expression in ancNif (arrow pointing downward). (B) Transcription fold change levels mapped to the tricarboxylic acid cycle. Gene products colored in gray are not significantly differentially expressed in ancNif (adjusted P < 0.05). The glyoxylate shunt, mediated by the icl gene product, is highlighted in orange.