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Resource allocation during the transition to diazotrophy in Klebsiella oxytoca

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Title: Resource allocation during the transition to diazotrophy in Klebsiella oxytoca
Authors: Waite, C
Lindstrom-Battle, A
Bennett, M
Carey, M
Kotta-Loizou, I
Hong, C
Buck, M
Schumacher, J
Item Type: Journal Article
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 fertilizers. 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 signalling 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 signalling 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.
Issue Date: Aug-2021
Date of Acceptance: 12-Jul-2021
URI: http://hdl.handle.net/10044/1/90597
DOI: 10.3389/fmicb.2021.718487
ISSN: 1664-302X
Publisher: Frontiers Media
Start Page: 1
End Page: 20
Journal / Book Title: Frontiers in Microbiology
Volume: 12
Copyright Statement: © 2021 Waite, Lindström Battle, Bennett, Carey, Hong, Kotta-Loizou, Buck and Schumacher. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Keywords: Klebsiella oxytoca
PII proteins
absolute protein quantification
diazotrophic bacteria: biological fixation of nitrogen
nif gene expression
nitrogen stress
resource allocation
0502 Environmental Science and Management
0503 Soil Sciences
0605 Microbiology
Publication Status: Published
Article Number: 718487
Online Publication Date: 2021-08-09
Appears in Collections:Department of Infectious Diseases
Faculty of Medicine
Faculty of Natural Sciences

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