Key Metabolites and Mechanistic Changes for Salt Tolerance in an Experimentally Evolved Sulfate-Reducing Bacterium, Desulfovibrio vulgaris

Abstract

Rapid genetic and phenotypic adaptation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough to salt stress was observed during experimental evolution. In order to identify key metabolites important for salt tolerance, a clone, ES10-5, which was isolated from population ES10 and allowed to experimentally evolve under salt stress for 5,000 generations, was analyzed and compared to clone ES9-11, which was isolated from population ES9 and had evolved under the same conditions for 1,200 generations. These two clones were chosen because they represented the best-adapted clones among six independently evolved populations. ES10-5 acquired new mutations in genes potentially involved in salt tolerance, in addition to the preexisting mutations and different mutations in the same genes as in ES9-11. Most basal abundance changes of metabolites and phospholipid fatty acids (PLFAs) were lower in ES10-5 than ES9-11, but an increase of glutamate and branched PLFA i17:1ω9c under high-salinity conditions was persistent. ES9-11 had decreased cell motility compared to the ancestor; in contrast, ES10-5 showed higher cell motility under both nonstress and high-salinity conditions. Both genotypes displayed better growth energy efficiencies than the ancestor under nonstress or high-salinity conditions. Consistently, ES10-5 did not display most of the basal transcriptional changes observed in ES9-11, but it showed increased expression of genes involved in glutamate biosynthesis, cation efflux, and energy metabolism under high salinity. These results demonstrated the role of glutamate as a key osmolyte and i17:1ω9c as the major PLFA for salt tolerance in D. vulgaris The mechanistic changes in evolved genotypes suggested that growth energy efficiency might be a key factor for selection.IMPORTANCE High salinity (e.g., elevated NaCl) is a stressor that affects many organisms. Salt tolerance, a complex trait involving multiple cellular pathways, is attractive for experimental evolutionary studies. Desulfovibrio vulgaris Hildenborough is a model sulfate-reducing bacterium (SRB) that is important in biogeochemical cycling of sulfur, carbon, and nitrogen, potentially for bio-corrosion, and for bioremediation of toxic heavy metals and radionuclides. The coexistence of SRB and high salinity in natural habitats and heavy metal-contaminated field sites laid the foundation for the study of salt adaptation of D. vulgaris Hildenborough with experimental evolution. Here, we analyzed a clone that evolved under salt stress for 5,000 generations and compared it to a clone evolved under the same condition for 1,200 generations. The results indicated the key roles of glutamate for osmoprotection and of i17:1ω9c for increasing membrane fluidity during salt adaptation. The findings provide valuable insights about the salt adaptation mechanism changes during long-term experimental evolution.

Keywords: Desulfovibrio vulgaris; PLFA; cell motility; energy efficiency; genomic mutations; organic solutes; transcriptomics.

FIG 1

Growth curves of colony-based salt-evolved D. vulgaris Hildenborough strains ES10-5 and ES9-11 and the ancestral strain (An) grown in LS4D, LS4D plus 250 mM NaCl, or LS4D plus 300 mM NaCl.

FIG 2

Accumulation of organic solutes in ES10-5, ES9-11, and the ancestral strain under nonstress conditions (LS4D) or high-salinity conditions (LS4D plus 300 mM NaCl). *, significant changes were induced under high-salinity conditions (LSD test, P < 0.05).

FIG 3

PLFA compositions in ES10-5, ES9-11, and the ancestral strain under nonstress conditions (LS4D) or high-salinity conditions (LS4D plus 300 mM NaCl). *, significant change induced by high salinity (LSD test, P < 0.05).

FIG 4

Physiological and transcriptional responses to high salinity for ES10-5, ES9-11, and the ancestral strain (An). (A) Cell motility. (B) Heat generated per cell under nonstress conditions (LS4D) and after transfer into high-salinity medium (LS4D plus 250 mM NaCl; transition). (C) DCA results for global transcriptional profiles. A subscript C after a strain designation indicates cells were cultured in LS4D; a subscript T after a strain designation indicates cells were cultured in LS4D plus 300 mM NaCl.