Development of modular expression across phylogenetically distinct diazotrophs

Abstract

Diazotrophic bacteria can reduce atmospheric nitrogen into ammonia enabling bioavailability of the essential element. Many diazotrophs closely associate with plant roots increasing nitrogen availability, acting as plant growth promoters. These associations have the potential to reduce the need for costly synthetic fertilizers if they could be engineered for agricultural applications. However, despite the importance of diazotrophic bacteria, genetic tools are poorly developed in a limited number of species, in turn narrowing the crops and root microbiomes that can be targeted. Here, we report optimized protocols and plasmids to manipulate phylogenetically diverse diazotrophs with the goal of enabling synthetic biology and genetic engineering. Three broad-host-range plasmids can be used across multiple diazotrophs, with the identification of one specific plasmid (containing origin of replication RK2 and a kanamycin resistance marker) showing the highest degree of compatibility across bacteria tested. We then demonstrated modular expression by testing seven promoters and eleven ribosomal binding sites using proxy fluorescent proteins. Finally, we tested four small molecule inducible systems to report expression in three diazotrophs and demonstrated genome editing in Klebsiella michiganensis M5al.

One-sentence summary: In this study, broad-host plasmids and synthetic genetic parts were leveraged to enable expression tools in a library of diazotrophic bacteria.

Keywords: biological nitrogen fixation; diazotroph; rhizosphere; synthetic biology.

Graphical Abstract

Fig. 1.

Graphical representation of phylogenetic distribution. From left to right: the relative phylum, class, and order of each diazotroph (phylogenetic tree not to scale) followed by strain, nitrogen fixation modality (BNF association) as either endophytic (associated with roots), free-living (independent of plants), or symbiotic (root nodule symbiosis), and the native host from which the strain was isolated.

Fig. 2.

Expression of sfGFP using Anderson Promoters. Schematic of the Anderson promoter library (A) highlighting nucleotide differences from wildtype promoter J23119 and the lowest expression promoter as assessed in E. coli J23103. Mean fluorescence intensity (MFI) in arbitrary units (AU) across selected diazotrophs in logarithmic scale (B–F). Promoters are organized from highest fluorescence to lowest (as reported in E. coli) for each diazotroph (left to right). Error bars represent standard deviation over three biological replicates. Full promoter sequences can be found in Supplementary Table S2.

Fig. 3.

Behavior of A. brasilense-optimized Ribosomal binding sites on gene expression across divergent diazotrophs. Representation of RBS plasmid library harboring mScarlet fluorescence protein (A) controlled by J23119 promoter and variable RBSs by altering nucleotide bases highlighted in red. Mean fluorescence intensity (MFI) in arbitrary units (AU) of mScarlet in logarithmic scale for selected diazotrophs (B–F). RBSs are organized by decreasing fluorescence in each diazotroph. Sequences 1 and 11 represent the RBS that have the highest and lowest predicted activity, respectively. Error bars represent standard deviation over three biological replicates. Dots denote one biological replicate. Full RBS sequences are available in Supplementary Table S2. Highest fluorescence normalized to a value of one reveals divergence of RBSs in different species (G).

Fig. 4.

Inducible promoter systems in diazotrophs. Mean fluorescence intensity (MFI) in arbitrary units (AU) of inducible promoter systems in H. seropedicae (A), K. michiganensis (B), and R. leguminosarum (C) plotted in linear scale. Small molecule inducers arabinose (Ara, squares), salicylic acid (Sal, triangles), and vanillic acid (Van, diamonds) were varied in concentration from 0.05 μm to 2000 μm. Error bars represent standard deviation over two biological replicates. Non-functional inducer systems are summarized in Supplementary Fig. S7.