Resource Allocation During the Transition to Diazotrophy in Klebsiella oxytoca
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 fixati...
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Published in | Frontiers in microbiology Vol. 12; p. 718487 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Frontiers Media S.A
09.08.2021
|
Subjects | |
Online Access | Get full text |
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Summary: | 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 N
2
. 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 (
nifK
1
–
1
203
), 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 Δ
nifLA
and
nifK
1
–
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 Δ
glnK
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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 These authors share senior authorship This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology Edited by: Harry Beller, U.S. Environmental Protection Agency (EPA), United States Reviewed by: Marcelo Bueno Batista, Department of Molecular Microbiology, John Innes Centre, United Kingdom; Luis M. Rubio, Center for Plant Biotechnology and Genomics, National Institute of Agricultural and Food Research and Technology, Spain; Fabio Pedrosa, Federal University of Paraná, Brazil |
ISSN: | 1664-302X 1664-302X |
DOI: | 10.3389/fmicb.2021.718487 |