Resource Allocation During the Transition to Diazotrophy in Klebsiella oxytoca

Christopher J Waite,Mark H Bennett,Anya Lindström Battle,Chun K Hong,Martin Buck,Matthew R Carey,Ioly Kotta-Loizou,Jörg Schumacher

doi:10.3389/fmicb.2021.718487

Abstract

Free-living nitrogen-fixing bacteria can improve growth yields of some non-leguminous plants and, if enhanced through bioengineering approaches, have the potential to address major nutrient imbalances in global crop production by supplementing inorganic nitrogen fertilisers. However, nitrogen fixation is a highly resource-costly adaptation and is de-repressed only in environments in which sources of reduced nitrogen are scarce. Here we investigate nitrogen fixation (nif) gene expression and nitrogen starvation response signaling in the model diazotroph Klebsiella oxytoca (Ko) M5a1 during ammonium depletion and the transition to growth on atmospheric N2. Exploratory RNA-sequencing revealed that over 50% of genes were differentially expressed under diazotrophic conditions, among which the nif genes are among the most highly expressed and highly upregulated. Isotopically labelled QconCAT standards were designed for multiplexed, absolute quantification of Nif and nitrogen-stress proteins via multiple reaction monitoring mass spectrometry (MRM-MS). Time-resolved Nif protein concentrations were indicative of bifurcation in the accumulation rates of nitrogenase subunits (NifHDK) and accessory proteins. We estimate that the nitrogenase may account for more than 40% of cell protein during diazotrophic growth and occupy approximately half the active ribosome complement. The concentrations of free amino acids in nitrogen-starved cells were insufficient to support the observed rates of Nif protein expression. Total Nif protein accumulation was reduced 10-fold when the NifK protein was truncated and nitrogenase catalysis lost (nifK1–1203), implying that reinvestment of de novo fixed nitrogen is essential for further nif expression and a complete diazotrophy transition. Several amino acids accumulated in non-fixing ΔnifLA and nifK1–1203 mutants, while the rest remained highly stable despite prolonged N starvation. Monitoring post-translational uridylylation of the PII-type signaling proteins GlnB and GlnK revealed distinct nitrogen regulatory roles in Ko M5a1. GlnK uridylylation was persistent throughout the diazotrophy transition while a ΔglnK mutant exhibited significantly reduced Nif expression and nitrogen fixation activity. Altogether, these findings highlight quantitatively the scale of resource allocation required to enable the nitrogen fixation adaptation to take place once underlying signaling processes are fulfilled. Our work also provides an omics-level framework with which to model nitrogen fixation in free-living diazotrophs and inform rational engineering strategies.

Highlights

Most organisms are unable to directly assimilate inert atmospheric dinitrogen (N2) and rely instead on the bioavailability of chemically reduced, or “fixed,” forms of nitrogen (N), such as ammonia, nitrate or amino acids
We supplemented Ko M5a1 with an initial ammonium salt concentration (0.5 mM NH4Cl) that enabled both nitrogen starvation and, subsequently, diazotrophic growth to be observed over the course of a day culture
Given the longstanding biotechnological interest in enhancing biological nitrogen fixation, it is remarkable that our understanding of diazotrophic bacteria has seldom progressed beyond a description of the various gene regulatory mechanisms governing nif gene expression to complete systems-level studies that integrate omics datasets and their interactions

Summary

Introduction

Most organisms are unable to directly assimilate inert atmospheric dinitrogen (N2) and rely instead on the bioavailability of chemically reduced, or “fixed,” forms of nitrogen (N), such as ammonia, nitrate or amino acids. Food security is fundamentally dependent on the uptake of fixed N by agricultural crops. Biological N fixation – the reduction of N2 to NH3 by specialised microorganisms – replenishes most of the fixed N in the biosphere and supports approximately 60% of the N required for human nutrition (Smil, 2001). As crop production must increase by up to 70% to support the estimated global food demand in 2050 (Hunter et al, 2017), agriculture is growing ever more reliant on the synthetically fixed and environmentally damaging N fertilisers produced by the industrial Haber–Bosch process. Modern biotechnological approaches offer great promise for expanding the capacity of biological N fixation and the development of more sustainable solutions for the delivery of N to plants (e.g., Rogers and Oldroyd, 2014; Batista and Dixon, 2019)

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