Experimental evolution reveals nitrate tolerance mechanisms in Desulfovibrio vulgaris

Abstract

Elevated nitrate in the environment inhibits sulfate reduction by important microorganisms of sulfate-reducing bacteria (SRB). Several SRB may respire nitrate to survive under elevated nitrate, but how SRB that lack nitrate reductase survive to elevated nitrate remains elusive. To understand nitrate adaptation mechanisms, we evolved 12 populations of a model SRB (i.e., Desulfovibrio vulgaris Hildenborough, DvH) under elevated NaNO3 for 1000 generations, analyzed growth and acquired mutations, and linked their genotypes with phenotypes. Nitrate-evolved (EN) populations significantly (p < 0.05) increased nitrate tolerance, and whole-genome resequencing identified 119 new mutations in 44 genes of 12 EN populations, among which six functional gene groups were discovered with high mutation frequencies at the population level. We observed a high frequency of nonsense or frameshift mutations in nitrosative stress response genes (NSR: DVU2543, DVU2547, and DVU2548), nitrogen regulatory protein C family genes (NRC: DVU2394-2396, DVU2402, and DVU2405), and nitrate cluster (DVU0246-0249 and DVU0251). Mutagenesis analysis confirmed that loss-of-functions of NRC and NSR increased nitrate tolerance. Also, functional gene groups involved in fatty acid synthesis, iron regulation, and two-component system (LytR/LytS) known to be responsive to multiple stresses, had a high frequency of missense mutations. Mutations in those gene groups could increase nitrate tolerance through regulating energy metabolism, barring entry of nitrate into cells, altering cell membrane characteristics, or conferring growth advantages at the stationary phase. This study advances our understanding of nitrate tolerance mechanisms and has important implications for linking genotypes with phenotypes in DvH.

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Fig. 1: Increased NaNO3 tolerance in EN populations compared to AN populations.
Fig. 2: Growth phenotype of DvH parental strain (JZ001) and marker exchange mutants (ME2543, ME2547 and ME2548) of nitrosative stress response genes (NSR).
Fig. 3: Growth phenotype of DvH parental strain (JZ001) and marker exchange mutants (ME2394, ME2395, ME2396, and ME2405) of nitrogen regulatory protein family C genes (NRC).
Fig. 4: A schematic representation of possible nitrate tolerance mechanisms in DvH.

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Acknowledgements

The experimental work of this study (October 2013 to June 2017) was largely supported by ENIGMA-Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Science Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, and Office of Biological & Environmental Research under contract number DE-AC02-05CH11231, the Office of the Vice President for Research at the University of Oklahoma (JZ), and the National Program on Key Basic Research Project of China (973 Program, No. 2015CB150505 to SC); the subsequent integration work (October 2018 to March 2020) of this study, including supplementary experiments, data analysis and manuscript writing, was mainly supported by the National Natural Science Foundation of China (Grant numbers 31770539 and 91951207 to ZH, grant number 31672262 to QY), and the Special Funds for Scientific and Technological Innovation in Guangdong Province (Grant number 2018A030310302 to BW).

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Wu, B., Liu, F., Zhou, A. et al. Experimental evolution reveals nitrate tolerance mechanisms in Desulfovibrio vulgaris. ISME J 14, 2862–2876 (2020). https://doi.org/10.1038/s41396-020-00753-5

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