Genetic Basis of Chromate Adaptation and the Role of the Pre-existing Genetic Divergence during an Experimental Evolution Study with Desulfovibrio vulgaris Populations

Abstract

Hexavalent chromium [Cr(VI)] is a common environmental pollutant. However, little is known about the genetic basis of microbial evolution under Cr(VI) stress and the influence of the prior evolution histories on the subsequent evolution under Cr(VI) stress. In this study, Desulfovibrio vulgaris Hildenborough (DvH), a model sulfate-reducing bacterium, was experimentally evolved for 600 generations. By evolving the replicate populations of three genetically diverse DvH clones, including ancestor (AN, without prior experimental evolution history), non-stress-evolved EC3-10, and salt stress-evolved ES9-11, the contributions of adaptation, chance, and pre-existing genetic divergence to the evolution under Cr(VI) stress were able to be dissected. Significantly decreased lag phases under Cr(VI) stress were observed in most evolved populations, while increased Cr(VI) reduction rates were primarily observed in populations evolved from EC3-10 and ES9-11. The pre-existing genetic divergence in the starting clones showed strong influences on the changes in lag phases, growth rates, and Cr(VI) reduction rates. Additionally, the genomic mutation spectra in populations evolved from different starting clones were significantly different. A total of 14 newly mutated genes obtained mutations in at least two evolved populations, suggesting their importance in Cr(VI) adaptation. An in-frame deletion mutation of one of these genes, the chromate transporter gene DVU0426, demonstrated that it played an important role in Cr(VI) tolerance. Overall, our study identified potential key functional genes for Cr(VI) tolerance and demonstrated the important role of pre-existing genetic divergence in evolution under Cr(VI) stress conditions. IMPORTANCE Chromium is one of the most common heavy metal pollutants of soil and groundwater. The potential of Desulfovibrio vulgaris Hildenborough in heavy metal bioremediation such as Cr(VI) reduction was reported previously; however, experimental evidence of key functional genes involved in Cr(VI) resistance are largely unknown. Given the genetic divergence of microbial populations in nature, knowledge on how this divergence affects the microbial adaptation to a new environment such as Cr(VI) stress is very limited. Taking advantage of our previous study, three groups of genetically diverse D. vulgaris Hildenborough populations with or without prior experimental evolution histories were propagated under Cr(VI) stress for 600 generations. Whole-population genome resequencing of the evolved populations revealed the genomic changes underlying the improved Cr(VI) tolerance. The strong influence of the pre-existing genetic divergence in the starting clones on evolution under Cr(VI) stress conditions was demonstrated at both phenotypic and genetic levels.

Keywords: Desulfovibrio vulgaris; chromate stress; experimental evolution; genetic background.

FIG 1

Schematic representation of the experimental design. Laboratory experimental evolution of DvH populations under Cr(VI) stress was initiated from three starting clones. The ancestor clone (AN) had no experimental evolution history. EC3-10 and ES9-11 were isolated from populations that had been propagated from AN under control or salt stress conditions for 1,200 generations, respectively (evolution history). Six replicates from each starting clone were evolved under Cr(VI) stress for 600 generations (new evolution).

FIG 2

Improved Cr(VI) tolerance and Cr(VI) reduction rate in salt (NaCl) stress-evolved clone ES9-11 compared to the control-evolved EC3-10 clone and AN. The growth rates (a) and lag phases (b) of the three starting clones in LS4D medium supplemented with 0.16 mM Cr(VI) are shown (t test, P < 0.01). (c) Cr(VI) reduction rates of EC3-10 and ES9-11 relative to AN at 20 min, 40 min, and 60 min after addition of Cr(VI) in the washed-cell experiments. *, P < 0.05 (t test). Error bars represent standard deviations for three biological replicates.

FIG 3

Cr(VI) resistance (lag phases [a] and growth rates [c]) and Cr(VI) reduction rates [60 min after addition of Cr(VI)] (e) of the Cr(VI) stress-evolved populations and contributions of the pre-existing genetic divergence, chance, and adaptation to the evolved mean lag phases (b), mean growth rates (d), and mean Cr(VI) reduction rates (f). Filled symbols in a, c, and e indicate significantly different values from the corresponding ancestor (t test, P < 0.05). Error bars represent the standard deviations for three biological replicates.

FIG 4

Analysis of the new mutations in the Cr(VI) stress-evolved populations. (a) Classification of the new mutations. SV, structural variant. (b) Detailed information of the 14 newly mutated genes found in at least two evolved populations. Genes labeled with stars were reported to have higher expression in DvH biofilms than in batch planktonic cells. (c) NMDS ordination of the combined gene level (77 genes) mutation frequencies of the new mutations (stress = 0.15). The frequencies of all the mutations in one gene were combined to represent the mutation frequency of this gene. Each symbol represents one population. (d) Categorization of the new mutations based on COG designations of the mutated genes. The full list of the COG annotations is in Table S1.

FIG 5

Cr(VI) resistance and Cr(VI) reduction ability of the DVU0426 mutants. (a) Predicted protein structures of DVU0426 in the parental strain JZ001 and evolved populations harboring DVU0426 mutations. The numbers indicate the positions of the amino acid residues. The blue boxes represent the chromate transporter domain ChrA. Red color indicates the predicted DVU0426 protein structure changes resulted from the mutations. The mutation frequencies are noted in blue. LMD, long mutant deletion; SMD, short mutant deletion. Growth curves of the parental strain JZ001 and the DVU0426 mutants in LS4D medium (b) or LS4D supplemented with 0.16 mM Cr(VI) (c) and their Cr(VI) reduction capability (d) tested with washed-cell experiments are shown. The error bars represent the standard deviations for three biological replicates.