Comparative Genomic Analysis Reveals the Distribution, Organization, and Evolution of Metal Resistance Genes in the Genus Acidithiobacillus

Abstract

Members of the genus Acidithiobacillus, which can adapt to extremely high concentrations of heavy metals, are universally found at acid mine drainage (AMD) sites. Here, we performed a comparative genomic analysis of 37 strains within the genus Acidithiobacillus to answer the untouched questions as to the mechanisms and the evolutionary history of metal resistance genes in Acidithiobacillus spp. The results showed that the evolutionary history of metal resistance genes in Acidithiobacillus spp. involved a combination of gene gains and losses, horizontal gene transfer (HGT), and gene duplication. Phylogenetic analyses revealed that metal resistance genes in Acidithiobacillus spp. were acquired by early HGT events from species that shared habitats with Acidithiobacillus spp., such as Acidihalobacter, Thiobacillus, Acidiferrobacter, and Thiomonas species. Multicopper oxidase genes involved in copper detoxification were lost in iron-oxidizing Acidithiobacillus ferridurans, Acidithiobacillus ferrivorans, and Acidithiobacillus ferrooxidans and were replaced by rusticyanin genes during evolution. In addition, widespread purifying selection and the predicted high expression levels emphasized the indispensable roles of metal resistance genes in the ability of Acidithiobacillus spp. to adapt to harsh environments. Altogether, the results suggested that Acidithiobacillus spp. recruited and consolidated additional novel functionalities during the adaption to challenging environments via HGT, gene duplication, and purifying selection. This study sheds light on the distribution, organization, functionality, and complex evolutionary history of metal resistance genes in Acidithiobacillus spp.IMPORTANCE Horizontal gene transfer (HGT), natural selection, and gene duplication are three main engines that drive the adaptive evolution of microbial genomes. Previous studies indicated that HGT was a main adaptive mechanism in acidophiles to cope with heavy-metal-rich environments. However, evidences of HGT in Acidithiobacillus species in response to challenging metal-rich environments and the mechanisms addressing how metal resistance genes originated and evolved in Acidithiobacillus are still lacking. The findings of this study revealed a fascinating phenomenon of putative cross-phylum HGT, suggesting that Acidithiobacillus spp. recruited and consolidated additional novel functionalities during the adaption to challenging environments via HGT, gene duplication, and purifying selection. Altogether, the insights gained in this study have improved our understanding of the metal resistance strategies of Acidithiobacillus spp.

Keywords: Acidithiobacillus spp.; comparative genomics; evolution; horizontal gene transfer; metal resistance.

FIG 1

Pangenome analysis of strains in the genus Acidithiobacillus. (A) Petal diagram of the pangenome. Each strain is represented by a colored oval. The center is the number of orthologous coding sequences shared by all strains (i.e., the core genome). Numbers in nonoverlapping portions of each oval show the numbers of CDSs unique to each strain. The total numbers of protein-coding genes within each genome are listed below the strain names. (B) Mathematical modeling of the pangenome and core genome of Acidithiobacillus. (C) Proportions of genes involved in heavy metal resistance in unique genes, accessory genome, core genome, and pangenome according BacMet database annotation.

FIG 2

Chronogram of the 37 Acidithiobacillus strains. The value near each internal branch is the estimated emerge time for that branch. Nodes with fossil record corrections are indicated with a red star.

FIG 3

Neighbor-joining (NJ) phylogenetic tree of the MerA protein sequences derived from Acidithiobacillus species strains and other representative species. Bootstrap values indicated at each node are based on a total of 1,000 bootstrap replicates. Branches representing group I mer clusters are in green, those representing group II mer clusters are in blue, and those representing group III mer clusters are in red and pink.

FIG 4

Neighbor-joining phylogenetic tree of concatenated ArsBRC protein sequences derived from Acidithiobacillus species strains and other representative species. Bootstrap values indicated at each node are based on a total of 1,000 bootstrap replicates. Branches representing ars clusters of Acidithiobacillus spp. are in blue.

FIG 5

Neighbor-joining phylogenetic tree of concatenated ArsAD protein sequences derived from Acidithiobacillus species strains and other representative species. Bootstrap values indicated at each node are based on a total of 1,000 bootstrap replicates. Branches representing ars clusters of Acidithiobacillus spp. are marked in red.

FIG 6

Neighbor-joining phylogenetic tree of concatenated CzcA, CzcB, and CzcC protein sequences derived from Acidithiobacillus species strains and other representative species. Bootstrap values indicated at each node are based on a total of 1,000 bootstrap replicates. Branches representing czcABC clusters are in green; czcBAC clusters formed in another group are in blue.

FIG 7

Distributions and ranges of selection pressures on different metal resistance genes of Acidithiobacillus spp.

FIG 8

Ranges of CAI values of different metal resistance genes of Acidithiobacillus spp. with Tu gene as a reference.