Taxonomically-linked growth phenotypes during arsenic stress among arsenic resistant bacteria isolated from soils overlying the Centralia coal seam fire

Abstract

Arsenic (As), a toxic element, has impacted life since early Earth. Thus, microorganisms have evolved many As resistance and tolerance mechanisms to improve their survival outcomes given As exposure. We isolated As resistant bacteria from Centralia, PA, the site of an underground coal seam fire that has been burning since 1962. From a 57.4°C soil collected from a vent above the fire, we isolated 25 unique aerobic As resistant bacterial strains spanning seven genera. We examined their diversity, resistance gene content, transformation abilities, inhibitory concentrations, and growth phenotypes. Although As concentrations were low at the time of soil collection (2.58 ppm), isolates had high minimum inhibitory concentrations (MICs) of arsenate and arsenite (>300 mM and 20 mM respectively), and most isolates were capable of arsenate reduction. We screened isolates (PCR and sequencing) using 12 published primer sets for six As resistance genes (AsRGs). Genes encoding arsenate reductase (arsC) and arsenite efflux pumps (arsB, ACR3(2)) were present, and phylogenetic incongruence between 16S rRNA genes and AsRGs provided evidence for horizontal gene transfer. A detailed investigation of differences in isolate growth phenotypes across As concentrations (lag time to exponential growth, maximum growth rate, and maximum OD590) showed a relationship with taxonomy, providing information that could help to predict an isolate's performance given As exposure in situ. Our results suggest that microbiological management and remediation of environmental As could be informed by taxonomically-linked As tolerance, potential for resistance gene transferability, and the rare biosphere.

Fig 1. Phylogenetic tree of 16S rRNA sequences from Centralia As resistant isolates.

Isolates from this study were compared with isolates from other studies that cultivated As resistant isolates from soil. (A) Actinobacteria, Proteobacteria, and Sphingobacteria. (B) Firmicutes. Scale bars indicate the percent difference in nucleotide sequence.

Fig 2. As resistance genotypes and phenotypes of isolated bacterial strains.

(A) Presence of AsRG from end-point PCR are indicated (+). MICs of (B) sodium arsenate and (C) arsenite. (D) Categorical range of arsenate reduced based on standard curve of known ratios of arsenate and arsenite.

Fig 3. Comparison of AsRG sequences and 16S rRNA gene sequences from As resistant isolates.

Maximum likelihood trees for AsRGs (left panel) (A) arsB, (B) ACR3(2), and (C) arsC are shown alongside trees of corresponding 16S rRNA genes (right panel). Incongruence is highlighted with grey lines between the two trees. Scale bars indicate the percent difference in nucleotide sequence. Bootstrap values greater than 50% are indicated at the corresponding node, and boxes are colored based on isolate genus.

Fig 4. Growth phenotypes of isolates in increasing concentrations of As.

Lag time (λ) and maximum growth rate (μ) of isolates in TSB50 with increasing concentrations of (A) arsenate and (B) arsenite normalized to growth without As.