Mixed waste contamination selects for a mobile genetic element population enriched in multiple heavy metal resistance genes.

Mobile genetic elements (MGEs) like plasmids, viruses, and transposable elements can provide fitness benefits to their hosts for survival in the presence of environmental stressors. Heavy metal resistance genes (HMRGs) are frequently observed on MGEs, suggesting that MGEs may be an important driver of adaptive evolution in environments contaminated with heavy metals. Here, we report the meta-mobilome of the heavy metal-contaminated regions of the Oak Ridge Reservation subsurface. This meta-mobilome was compared with one derived from samples collected from unimpacted regions of the Oak Ridge Reservation subsurface. We assembled 1615 unique circularized DNA elements that we propose to be MGEs. The circular elements from the highly contaminated subsurface were enriched in HMRG clusters relative to those from the nearby unimpacted regions. Additionally, we found that these HMRGs were associated with Gamma and Betaproteobacteria hosts in the contaminated subsurface and potentially facilitate the persistence and dominance of these taxa in this region. Finally, the HMRGs were associated with conjugative elements, suggesting their potential for future lateral transfer. We demonstrate how our understanding of MGE ecology, evolution, and function can be enhanced through the genomic context provided by completed MGE assemblies.

Genomic and environmental controls on Castellaniella biogeography in an anthropogenically disturbed subsurface.

Castellaniella species have been isolated from a variety of mixed-waste environments including the nitrate and multiple metal-contaminated subsurface at the Oak Ridge Reservation (ORR). Previous studies examining microbial community composition and nitrate removal at ORR during biostimulation efforts reported increased abundances of members of the Castellaniella genus concurrent with increased denitrification rates. Thus, we asked how genomic and abiotic factors control the Castellaniella biogeography at the site to understand how these factors may influence nitrate transformation in an anthropogenically impacted setting. We report the isolation and characterization of several Castellaniella strains from the ORR subsurface. Five of these isolates match at 100% identity (at the 16S rRNA gene V4 region) to two Castellaniella amplicon sequence variants (ASVs), ASV1 and ASV2, that have persisted in the ORR subsurface for at least 2 decades. However, ASV2 has consistently higher relative abundance in samples taken from the site and was also the dominant blooming denitrifier population during a prior biostimulation effort. We found that the ASV2 representative strain has greater resistance to mixed metal stress than the ASV1 representative strains. We attribute this resistance, in part, to the large number of unique heavy metal resistance genes identified on a genomic island in the ASV2 representative genome. Additionally, we suggest that the relatively lower fitness of ASV1 may be connected to the loss of the nitrous oxide reductase (nos) operon (and associated nitrous oxide reductase activity) due to the insertion at this genomic locus of a mobile genetic element carrying copper resistance genes. This study demonstrates the value of integrating genomic, environmental, and phenotypic data to characterize the biogeography of key microorganisms in contaminated sites.

Mixed nitrate and metal contamination influences operational speciation of toxic and essential elements.

Environmental contamination constrains microbial communities impacting diversity and total metabolic activity. The former S-3 Ponds contamination site at Oak Ridge Reservation (ORR), TN, has elevated concentrations of nitric acid and multiple metals from decades of processing nuclear material. To determine the nature of the metal contamination in the sediment, a three-step sequential chemical extraction (BCR) was performed on sediment segments from a core located upgradient (EB271, non-contaminated) and one downgradient (EB106, contaminated) of the S-3 Ponds. The resulting exchangeable, reducing, and oxidizing fractions were analyzed for 18 different elements. Comparison of the two cores revealed changes in operational speciation for several elements caused by the contamination. Those present from the S-3 Ponds, including Al, U, Co, Cu, Ni, and Cd, were not only elevated in concentration in the EB106 core but were also operationally more available with increased mobility in the acidic environment. Other elements, including Mg, Ca, P, V, As, and Mo, were less operationally available in EB106 having decreased concentrations in the exchangeable fraction. The bioavailability of essential macro nutrients Mg, Ca, and P from the two types of sediment was determined using three metal-tolerant bacteria previously isolated from ORR. Mg and Ca were available from both sediments for all three strains; however, P was not bioavailable from either sediment for any strain. The decreased operational speciation of P in contaminated ORR sediment may increase the dependence of the microbial community on other pools of P or select for microorganisms with increased P scavenging capabilities. Hence, the microbial community at the former S-3 Ponds contamination site may be constrained not only by increased toxic metal concentrations but also by the availability of essential elements, including P.

Mixed heavy metal stress induces global iron starvation response.

Multiple heavy metal contamination is an increasingly common global problem. Heavy metals have the potential to disrupt microbially mediated biogeochemical cycling. However, systems-level studies on the effects of combinations of heavy metals on bacteria are lacking. For this study, we focused on the Oak Ridge Reservation (ORR; Oak Ridge, TN, USA) subsurface which is contaminated with several heavy metals and high concentrations of nitrate. Using a native Bacillus cereus isolate that represents a dominant species at this site, we assessed the combined impact of eight metal contaminants, all at site-relevant concentrations, on cell processes through an integrated multi-omics approach that included discovery proteomics, targeted metabolomics, and targeted gene-expression profiling. The combination of eight metals impacted cell physiology in a manner that could not have been predicted from summing phenotypic responses to the individual metals. Exposure to the metal mixture elicited a global iron starvation response not observed during individual metal exposures. This disruption of iron homeostasis resulted in decreased activity of the iron-cofactor-containing nitrate and nitrite reductases, both of which are important in biological nitrate removal at the site. We propose that the combinatorial effects of simultaneous exposure to multiple heavy metals is an underappreciated yet significant form of cell stress in the environment with the potential to disrupt global nutrient cycles and to impede bioremediation efforts at mixed waste sites. Our work underscores the need to shift from single- to multi-metal studies for assessing and predicting the impacts of complex contaminants on microbial systems.

Ecophysiological and genomic analyses of a representative isolate of highly abundant Bacillus cereus strains in contaminated subsurface sediments.

Bacillus cereus strain CPT56D-587-MTF (CPTF) was isolated from the highly contaminated Oak Ridge Reservation (ORR) subsurface. This site is contaminated with high levels of nitric acid and multiple heavy metals. Amplicon sequencing of the 16S rRNA genes (V4 region) in sediment from this area revealed an amplicon sequence variant (ASV) with 100% identity to the CPTF 16S rRNA sequence. Notably, this CPTF-matching ASV had the highest relative abundance in this community survey, with a median relative abundance of 3.77% and comprised 20%-40% of reads in some samples. Pangenomic analysis revealed that strain CPTF has expanded genomic content compared to other B. cereus species-largely due to plasmid acquisition and expansion of transposable elements. This suggests that these features are important for rapid adaptation to native environmental stressors. We connected genotype to phenotype in the context of the unique geochemistry of the site. These analyses revealed that certain genes (e.g. nitrate reductase, heavy metal efflux pumps) that allow this strain to successfully occupy the geochemically heterogenous microniches of its native site are characteristic of the B. cereus species while others such as acid tolerance are mobile genetic element associated and are generally unique to strain CPTF.

Anoxic photochemical weathering of pyrite on Archean continents.

Sulfur is an essential element of life that is assimilated by Earth's biosphere through the chemical breakdown of pyrite. On the early Earth, pyrite weathering by atmospheric oxygen was severely limited, and low marine sulfate concentrations persisted for much of the Archean eon. Here, we show an anoxic photochemical mechanism of pyrite weathering that could have provided substantial amounts of sulfate to the oceans as continents formed in the late Archean. Pyrite grains suspended in anoxic ferrous iron solutions produced millimolar sulfate concentrations when irradiated with ultraviolet light. The Fe2+(aq) was photooxidized, which, in turn, led to the chemical oxidation of pyritic sulfur. Additional experiments conducted with 2.68 Ga shale demonstrated that photochemically derived ferric iron oxidizes and dissolves sedimentary pyrite during chemical weathering. The results suggest that before the rise of atmospheric oxygen, oxidative pyrite weathering on Archean continents was controlled by the exposure of land to sunlight.

Complete Genome Sequence of Bacillus cereus Strain CPT56D-587-MTF, Isolated from a Nitrate- and Metal-Contaminated Subsurface Environment.

Bacillus cereus strain CPT56D-587-MTF was isolated from nitrate- and toxic metal-contaminated subsurface sediment at the Oak Ridge Reservation (ORR) (Oak Ridge, TN, USA). Here, we report the complete genome sequence of this strain to provide genomic insight into its strategies for survival at this mixed-waste site.

Tellurite Adsorption onto Bacterial Surfaces.

Tellurium (Te) is an emerging contaminant and its chemical transformation in the environment is strongly influenced by microbial processes. In this study, we investigated the adsorption of tellurite [Te(IV), TeO32-] onto the common soil bacterium Bacillus subtilis. Thiol-blocking experiments were carried out to investigate the role of cell surface sulfhydryl sites in tellurite binding, and extended X-ray absorption fine structure (EXAFS) spectroscopy was performed to determine the chemical speciation of the adsorbed tellurite. The results indicate that tellurite reacts with sulfhydryl functional groups in the extracellular polymeric substances (EPS) produced by B. subtilis. Upon binding to sulfhydryl sites in the EPS, the Te changes from Te-O bonds to Te-S coordination. Further analysis of the surface-associated molecules shows that the EPS of B. subtilis contain proteins. Removal of the proteinaceous EPS dramatically decreases tellurite adsorption and the sulfhydryl surface site concentration. These findings indicate that sulfhydryl binding in EPS plays a key role in tellurite adsorption on bacterial surfaces.

The role of cysteine in tellurate reduction and toxicity.

The tellurium oxyanion tellurate is toxic to living organisms even at low concentrations; however, its mechanism of toxicity is poorly understood. Here, we show that exposure of Escherichia coli K-12 to tellurate results in reduction to elemental tellurium (Te[0]) and the formation of intracellular reactive oxygen species (ROS). Toxicity assays performed with E. coli indicated that pre-oxidation of the intracellular thiol pools increases cellular resistance to tellurate-suggesting that intracellular thiols are important in tellurate toxicity. X-ray absorption spectroscopy experiments demonstrated that cysteine reduces tellurate to elemental tellurium. This redox reaction was found to generate superoxide anions. These results indicate that tellurate reduction to Te(0) by cysteine is a source of ROS in the cytoplasm of tellurate-exposed cells.

Extracellular sulfite is protective against reactive oxygen species and antibiotic stress in Shewanella oneidensis MR-1.

In this study, we investigated the extracellular reactive sulfur species produced by Shewanella oneidensis MR-1 during growth. The results showed that sulfite is the major extracellular sulfur metabolite released to the growth medium under both aerobic and anaerobic growth conditions. Exogenous sulfite at physiological concentrations protected S. oneidensis MR-1 from hydrogen peroxide toxicity and enhanced tolerance to the beta-lactam antibiotics cefazolin, meropenem, doripenem and ertapenem. These findings suggest that the release of extracellular sulfite is a bacterial defence mechanism that plays a role in the mitigation of environmental stress.