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

<div><p>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 (<i>arsC</i>) and arsenite efflux pumps (<i>arsB</i>, <i>ACR3(2)</i>) 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 <i>in situ</i>. 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.</p></div

Ecological selection for small microbial genomes along a temperate-to-thermal soil gradient

Small bacterial and archaeal genomes provide insights into the minimal requirements for life(1) and are phylogenetically widespread(2). However, the precise environmental pressures that constrain genome size in free-living microorganisms are unknown. A study including isolates has shown that thermophiles and other bacteria with high optimum growth temperatures often have small genomes(3). It is unclear whether this relationship extends generally to microorganisms in nature(4,5) and more specifically to microorganisms that inhabit complex and highly variable environments, such as soils(3,6,7). To understand the genomic traits of thermally adapted microorganisms, here we investigated metagenomes from a 45 °C gradient of temperate-to-thermal soils that lie over the ongoing Centralia, Pennsylvania (USA) coal-seam fire. We found that hot soils harboured distinct communities with small genomes and small cell sizes relative to those in ambient soils. Hot soils notably lacked genes that encode known two-component regulatory systems, and antimicrobial production and resistance genes. Our results provide field evidence for the inverse relationship between microbial genome size and temperature in a diverse, free-living community over a wide range of temperatures that support microbial life. (1.) Hutchison, C. A. et al. Design and synthesis of a minimal bacterial genome. Science 351, aad6253 (2016). (2.) Hug, L. A. et al. A new view of the tree of life. Nat. Microbiol. 1, 16048 (2016). (3.) Sabath, N., Ferrada, E., Barve, A. & Wagner, A. Growth temperature and genome size in bacteria are negatively correlated, suggesting genomic streamlining during thermal adaptation. Genome Biol. Evol. 5, 966–977 (2013). (4.) Huete-Stauffer, T. M., Arandia-Gorostidi, N., Alonso-Sáez, L. & Morán, X. A. G. Experimental warming decreases the average size and nucleic acid content of marine bacterial communities. Front. Microbiol. 7, 730 (2016). (5.) Swan, B. K. et al. Prevalent genome streamlining and latitudinal divergence of planktonic bacteria in the surface ocean. Proc. Natl Acad. Sci. USA 110, 11463–11468 (2013). (6.) Brewer, T. E., Handley, K. M., Carini, P., Gilbert, J. A. & Fierer, N. Genome reduction in an abundant and ubiquitous soil bacterium ‘Candidatus Udaeobacter copiosus’. Nat. Microbiol. 2, 16198 (2016). (7.) Giovannoni, S. J., Thrash, J. C. & Temperton, B. Implications of streamlining theory for microbial ecology. ISME J. 8, 1553–1565 (2014)

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

<p>Maximum likelihood trees for AsRGs (left panel) (A) <i>arsB</i>, (B) <i>ACR3(2)</i>, and (C) <i>arsC</i> 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.</p

Relative abundance of isolate 16S rRNA gene sequences from our amplicon survey of the same soil.

<p>Relative abundance of isolate 16S rRNA gene sequences from our amplicon survey of the same soil.</p

As resistance genotypes and phenotypes of isolated bacterial strains.

<p>(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.</p

Growth phenotypes of isolates in increasing concentrations of As.

<p>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.</p

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

<p>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.</p

Data of enzymatic activities of the electron transport chain and ATP synthase complexes in mouse hepatoma cells following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

Abstract2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most widely studied ligand of the aryl hydrocarbon receptor (AHR). The AHR-dependent TCDD-induced mitochondrial hyperpolarization (Tappenden et al., 2011) [1] and reduced oxygen consumption rate of intact mouse hepatoma cells (Huang et al., in press) [2] in the previous studies suggest that these alterations can be related to enzymatic activities of the electron transport chain (ETC) and ATP synthase in oxidative phosphorylation (OXPHOS) system. Here, we evaluated the activity of each complex in the OXPHOS system using in vitro enzymatic assays. The calculated enzymatic activity of each complex was normalized against the activity of citrate synthase. To combine each value from an independent experiment, normalized enzyme activities from cells exposed to TCDD were converted to fold changes via comparison to the activity relative to time-matched vehicle control. The averaged fold change for each treatment suggests more replicates are needed in order to clearly evaluate a difference between treatments